Methods, pharmaceutical compositions and articles of manufacture for administering therapeutic cells to the animal central nervous system

ABSTRACT

Methods and compositions for preventing and treating the damaged and/or degenerating CNS experiencing loss or death of CNS cells. Various embodiments of the invention transport a therapeutically effective amount of, inter alia at least one therapeutic cell to the CNS by intranasal application to the upper-third of the nasal cavity, thereby bypassing the blood-brain barrier. A pharmaceutical composition according to the invention may comprise at least one therapeutic cell, at least one delivery-enhancement agent, at least one antibiotic, at least one regulatory factor and/or at least one immunosuppressive agent, wherein the composition is delivered to the upper-third of the nasal cavity. The therapeutic cells, once delivered to the CNS, migrate preferentially to the area of damage or degeneration or injury.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of patent application Ser. No.13/647,066, filed Oct. 8, 2012, which is a continuation of applicationSer. No. 12/109,066, filed Apr. 24, 2008, now U.S. Pat. No. 8,283,160which claims the benefit of provisional patent application Ser. No.60/971,284, filed Sep. 11, 2007, the entire contents of which are herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to methods and pharmaceuticalcompositions for administering therapeutic cells to the upper third ofthe nasal cavity of a mammal, thereby enabling the therapeutic cells tobypass the blood-brain barrier to prevent and/or treat the mammal'sdamaged and/or degenerating and/or injured central nervous system.

2. Description of the Related Art

Many neurological conditions result from damage to or the loss, i.e.,death, of certain cell populations from the central nervous systemthrough aging, disease or Injury. The cells damaged or destroyed inthese conditions are not intrinsically replaced, thus the centralnervous system is damaged and/or degenerating with resulting loss offunction. Recent evidence demonstrates that neuronal replacement andpartial reconstruction of neuronal circuitry is possible via celltransplantation therapies. Much of the initial work in the field usedfetal-cell therapies. More recently, however, it has become evident thatthe developing and even the adult mammalian nervous system contains apopulation of undifferentiated, multipotent, neural stem cells thatdisplay plastic properties that are advantageous for the design of moreeffective neural regenerative strategies for many of these neurologicalconditions.

The neurological conditions, diseases and/or injuries resulting indamaged and/or degenerating CNS, i.e., cell death, comprise Alzheimer'sdisease, mild cognitive impairment, age-associated memory impairment,Parkinson's disease, cerebrovascular disease including stroke,Creutzfeldt-Jakob disease, familial amyotrophic lateral sclerosis,lewy-body dementia, atherosclerosis, schizophrenia, autism, tardivedyskinesia, multiple sclerosis, seizure disorders, Wilson's disease,progressive supranuclear palsy, Hallervorden-Spatz syndrome, multisystematrophy, Huntington's disease, familial basal ganglia degeneration,Down's syndrome, cataracts, haemochromatosis, thalassemia, cerebralhemorrhage, subarachnoic hemorrhage, head injury, and spinal cordinjury. Moreover, certain medical procedures, for example coronaryartery bypass graft (CABG) surgery, are associated with neurologicalcomplications that result in damage and/or degeneration of the centralnervous system and concomitant cell death. In the case of CABG, thesurgery is performed on more than 800,000 patients worldwide each year.Many of the CABG procedures performed are associated with neurologicalcomplications. These complications range from stroke in up to 16% of thepatients to general cognitive decline with 50% of patients havingimpairment post-surgery and with progressive decline occurring in somepatients over the next five years. In addition, physical and behavioralimpairment manifest in some CABG patients. Newman M F et al., N. Eng. J.Med. 344:395-402 (2001); Brillman J., Neurol. Clin. 11:475-495 (1993);and Seines, O. A., Ann. Thorac. Surg. 67:1669-1676 (1999) areinstructive.

Neural stem cells have been demonstrated to replace lost and dying cellsand lost neural circuits in the damaged and/or degenerating CNS in cellreplacement therapies. For instance, treatment of mice with MPTP, a drugthat selectively destroys dopaminergic cells in the brain stem, followedby grafting with a neural stem cell population, resulted in areconstituted dopaminergic cell population composed of both donor andhost cells. Similar studies in mice using a hypoxia-ischemic braininjury model showed transplantation of neural stem cells enhanced therecovery of the damaged system (Park et al. (1999) J. Neurotrauma16:675-687 and Park et al. (1997) Soc. Neurosci. Abst. 23:346). Inpatients with stroke, the transplantation of cells from a human neuronalcell line showed improvement of neurological function. (Kondziolka D.,et al., (2000) “Transplantation of cultured human neuronal cells forpatients with stroke”. Neurology. 55:565-9). In a mouse model ofAlzheimer's disease, the transplantation of neural stem cells into theprefrontal and parietal cortices dramatically alleviated the cholinergicdeficits and recent memory disruption associated with AD. (Wang, Q., etal., (2006) “Neural stem cells transplantation in cortex in a mousemodel of Alzheimer's disease. J Med Invest., 53:61-9).

Further, in Parkinson's disease, the neurons that degenerate in themammalian central nervous system comprise the dopaminergic neurons ofthe substantia nigra. Current cell replacement strategies for patientswith advanced Parkinson's disease comprise intrastriatal grafts ofnigral dopaminergic neurons from 6- to 9-week-old human embryos.Clinical improvements develop gradually over the first 6-24 months aftertransplantation (Olanow et al. (1996) Trends Neurosci. 19:102-109 andLindvall et al. (1999) Mov. Disord. 14:201-205). It has been shown thatstem cell transplants of different origin, e.g., hematopoietic,embryonic, result in several clinical benefits in patients with severeParkinson's disease. (Freed, C R, et al. (Transplantation of embryonicdopamine neurons for severe Parkinson's disease. N Engl J Med 2001;344:710-719).

Similar benefits were realized with progressive multiple sclerosispatients. (Ni X S, et al., (2006) “Autologous hematopoietic stem celltransplantation for progressive multiple sclerosis: report of efficacyand safety at three yr of follow up in 21 patients” Clin Transplant.20:485-9) (further suggesting that MS treatment should combineimmunomodulation with neuroprotective modulaties such as cell-basedtherapy to achieve maximal clinical benefit).

Further, the first study of human fetus-to-adult striataltransplantation has been performed in three nondemented patients withmoderately advanced Huntington disease. Magnetic resonance imagingevaluation at 1 year documented graft survival and growth withoutdisplacement of surrounding tissue. All patients improved on somemeasure of cognitive function. (Kopyov et al. (1998) J. Exp. Neurol.149:97-108). See also, Date et al. (1997) J. Exp. Neurol. 147:10-17.

Each of the known models and methods for therapeutic cell-basedtherapies require surgical intervention, i.e., transplantation, ofneural stem cells using invasive grafting techniques and/or systemicdelivery methods that do not target the damaged areas of the centralnervous system. It would be highly advantageous to provide a method,pharmaceutical composition and/or article of manufacture or kit thatwould provide therapeutic cells, including but not limited to neuralstem cells, in a non-invasive and highly targeted manner.

For example, it would be advantageous to deliver such therapeutic cellsto the degenerating central nervous system in such a way as to avoidsystemic exposure. No known method or pharmaceutical compositioncurrently provides such advantages. The present invention provides theseadvantages by applying the therapeutic cells to the upper third of thenasal cavity, thereby bypassing the blood-brain barrier andadministering the therapeutic cells and other compounds directly to thecentral nervous system.

Certain embodiments of the present invention comprise nasal and/ormucosal antibiotics to assist in protecting the subject patient fromnasal bacteria migrating along the neural pathway followed by theapplied therapeutic cells and/or pharmaceutical compound. Suchantibiotics are well known as applied topically, but none areadministered as a pretreatment, co-treatment and/or post-treatment,either systemically and/or intranasally, in conjunction with intranasalapplication of therapeutic cells and/or pharmaceutical compound.

For example, in one study, mupirocin smeared inside the nose cutinfection rates in half or better Staphylococcus aureus is a widelydistributed germ that normally resides in the nostrils of an estimated25 to 30 percent of all hospitalized patients without causing harm. Butthis bacteria can contaminate surgical sites, causing severe and oftendeadly infections, especially in people with weakened immune systems.

Another study found that nasal xylitol, an over the counter remedy soldin health food stores, can reduce nasal bacteria and their ability tohold onto and infect cells in the nasal mucosa. Still other studies havefound that defensins, a natural antibiotic found in mucosa in the human,can protect against bacterial infection and enhance immune protectivefunction. Mammalian defensins are small, cationic, antimicrobialpeptides encoded by the host that are considered to be importantantibiotic-like effectors of innate immunity. By using chemokinereceptors on dendritic cells and T cells, defensins might alsocontribute to the regulation of host adaptive immunity against microbialinvasion. Defensins have considerable immunological adjuvant activityand linkage of beta-defensins or selected chemokines to an idiotypiclymphoma antigen has yielded potent antitumor vaccines. The functionaloverlap between defensins and chemokines is reinforced by reports thatsome chemokines have antimicrobial activities. Although showingsimilarity in activity and overall tertiary structure, the evolutionaryrelationship between defensins and chemokines remains to be determined.(De Yang, et al., Mammalian defensins in immunity: more than justmicrobicidal. Trends Immunol. 2002 June; 23 (6):291-6 12072367).

Moreover, it is well known that regulatory agents comprising trophic andgrowth factors such as erythropoietin (EPO), brain-derived neurotrophicfactor (BDNF), nerve growth factor (NGF), fibroblast growth factor (FGF)and epidermal growth factor (EGF) play a crucial role in in-vitro andin-vivo survival and differentiation of stem cells (Erickson et al.,Roles of insulin and transferrin in neural progenitor survival andproliferation. J Neurosci Res. 2008 Feb. 21; Bossolasco et al.,Neuro-glial differentiation of human bone marrow stem cells in vitro.Exp Neural, 2005 June; 193(2):312-25). The better survival of surgicallytransplanted cells was shown in the case of simultaneous application ofEPO (Kanaan et al., Exogenous erythropoietin provides neuroprotection ofgrafted dopamine neurons in a rodent model of Parkinson's disease. BrainRes. 2006 Jan. 12; 1068(1):221-9). However, it is not known to introducesuch regulatory factors or agents in conjunction with the intranasalapplication of therapeutic cells and/or pharmaceutical compositionsthereof, to the upper third of the nasal cavity, thus bypassing theblood-brain barrier.

In addition, it is well known that regulatory agents comprising variousgrowth factors including insulin-like growth factor-I (IGF-I), nervegrowth factor (NGF), and basic fibroblast growth factor (bFGF), regulatethe survival and differentiation of nerve cells during the developmentof the peripheral and central nervous systems. Regulatory agents such asneurotrophins are also required for nerve growth during development(Tucker et al. (2001) Nature Neurosci, 4:29-37). In the mature nervoussystem, these trophic factors maintain the morphologic and neurochemicalcharacteristics of nerve cells and strengthen functionally activesynaptic connections. Such regulatory factors find use in enhancing themethods of cell-replacement therapies according to the presentinvention.

For instance, bFGF enhances survival and growth of neurons in vitro.Further, bFGF produces a potent growth promoting effect on implantedneurons in vivo when the implanted neurons are genetically engineered toexpress the bFGF (Takayama et al. (1995) Nat. Med. 1:53-8). In addition,implantation of polymer-based bioactive rods that secrete epidermalgrowth factor and bFGF into transplanted fetal ventral mesencyphalictissue result in both improved functional characteristics and enhancedcell survival (Tornquvist et al. (2000) Exp. Neurol. 164:130-138).

Nerve growth factor (NGF) has also been shown to influence graftedtissue in the CNS. For example, ChAT activity, an assay indicative ofcholinergic cell activity, was elevated significantly in cholinergicneurons that were transplanted into brain tissue that contained anNGF-releasing pellet adjacent to the grafted cells (Mahoney et al.(1999) Med. Sci. 96:4536-4539). IGF-I has also been shown to promotedifferentiation of post-mitotic mammalian CNS neuronal stem cells and toinfluence apoptosis of human erythroid progenitor cells. See, forexample, Arsenijevic et al. (1998) J. Neurosci. 18:2118-2128; Tanigachiet al. (1997) Blood 90:2244-2252; Reboarcet et al. (1996) J. Biol.Reprod. 55:1119-1125; Muta et al. (1994) J. Clin. Invest. 94:34-43; and,Muta et al. (1993) J. Cell. Phys. 156:264-271. Additionally, it has beenshown that certain growth associated proteins, such as, GAP-43 andCAP-23 act to promote regeneration of injured axons and may supportregeneration in the spinal cord and CNS. See, for example, Bomze et al.(2001) Nature Neurosci. 4:38-43 and Woolf et al. (2001) Nature Neurosci.4:7-9.

Administration of regulatory agents as a means of improving the clinicaloutcome of a mammal having undergone a neural regenerative, i.e.,therapeutic cell-based strategy has, however, been meet with difficulty.Generally, these agents cannot be administered systemically.Furthermore, many of these regulatory agents do not cross theblood-brain barrier efficiently. Intracerebroventricular administration,while possibly an effective method for delivering regulatory agents, isan invasive technique that is not preferred in a clinical setting.Implantation of polymers containing regulatory agents is also invasiveand is further limited by the relatively small radius surrounding thepolymer implant in which the regulatory agent is capable of eliciting aneffect. Additionally, while genetic engineering of the transplantedcells to express regulatory agents has been performed, stabletransfection and survival of the cells following implantation continuesto be problematic.

The present invention provides solutions for, inter alia, theseproblems.

SUMMARY OF THE INVENTION

Given the situation described above there is a need for a method forefficient and non-invasive delivery of therapeutic cells and/orpharmaceutical compositions to the damaged and/or degenerating centralnervous system.

The present invention is directed to, inter alia, the prevention and/ortreatment of the damaged and/or degenerating central nervous system dueto a disease or other condition that causes the loss or death of CNScells. Specifically, the present invention provides a method,pharmaceutical composition and article of manufacture for transporting atherapeutically effective amount of at least one therapeutic cell to theCNS by intranasal application to the upper third of the nasal cavity,thereby bypassing the blood-brain barrier and avoiding unwanted systemicexposure as well as invasive delivery methods.

Various embodiments of the present invention comprise intranasalprevention, pretreatment, post-treatment and/or as a component of thepharmaceutical composition comprising therapeutic cells of atherapeutically effective amount of a delivery-enhancement agent(s) toenhance delivery of the therapeutic cell(s) to the CNS. Still otherembodiments comprise at least one antibiotic applied intranasally and/orsystemically as a pretreatment, a co-treatment (either administeredsimultaneously or as a component of the therapeutic compositioncomprising therapeutic cells) and/or a post-treatment device to protectthe patient during therapeutic cell therapy. Still other embodimentscomprise administering a therapeutically effective amount of at leastone regulatory agent to the upper third of the mammalian nasal cavity asa pretreatment, post-treatment and/or as part of the pharmaceuticalcomposition comprising the therapeutic cells. Still other embodimentscomprise at least one immunosuppressive agent applied intranasallyand/or systemically as a pretreatment, a co-treatment (eitheradministered simultaneously or as a component of the therapeuticcomposition comprising therapeutic cells) and/or a post-treatment deviceto enhance the viability of therapeutic cells in vivo during therapeuticcell therapy. The present invention finds use in improving the clinicaloutcome of a mammal having undergone a neural regenerative strategycomprising the bypassing of the blood-brain barrier of therapeutic cellstransported directly into the CNS of the mammal.

Various embodiments of the invention relate to methods andpharmaceutical compositions for preventing and treating neurologicaldamage and degeneration, i.e., cell loss and death within the CNS andthe resulting effects, including but not limited to treating memory lossand improving memory loss, due to cerebral ischemia and/orneurodegeneration for patients at risk for, or diagnosed with, certainmedical conditions such as Alzheimer's disease, mild cognitiveimpairment, age-associated memory impairment, Parkinson's disease,cerebrovascular disease including stroke, Creutzfeldt-Jakob disease,familial amyotrophic lateral sclerosis, lewy-body dementia,atherosclerosis, schizophrenia, autism, tardive dyskinesia, multiplesclerosis, seizure disorders, Wilson's disease, progressive supranuclearpalsy, Hallervorden-Spatz syndrome, multisystem atrophy, Huntington'sdisease, familial basal ganglia degeneration, Down's syndrome,cataracts, haemochromatosis, thalassemia, cerebral hemorrhage,subarachnoid hemorrhage, head injury, spinal cord injury and metabolicdisorders affecting the CNS.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “central nervous system” (CNS) refers to the brain andspinal cord and associated tissues.

As used herein, “neurological disorders and diseases of the CNS” refersto brain diseases and conditions that comprise ischemia, i.e., cerebralischemia, ischemia, stroke, neurodegeneration, neurologicalcomplications arising from such as Alzheimer's disease, Parkinson'sdisease, Wilson's disease, Lewy body dementia, multiple sclerosis,seizure disorders, cerebellar ataxia, progressive supranuclear palsy,amyotrophic lateral sclerosis, autism, affective disorders, anxietydisorders, metabolic disorders that affect the CNS, and/orschizophrenia; cell damage; nerve damage from cerebrovascular disorderssuch as stroke in the brain or spinal cord, from CNS infectionsincluding meningitis and HIV, from tumors of the brain and spinal cord,prion diseases, and CNS disorders resulting from ordinary aging (e.g.,anosmia), head and/or brain injury, or spinal cord injury and any othermedical diseases and conditions mentioned herein with neurological cellloss, damage and/or degeneration.

An “effective amount” of cells and/or agent is an amount sufficient toprevent, treat, reduce and/or ameliorate the symptoms and/or underlyingcauses of any of the above disorders or diseases. In some instances, an“effective amount” is sufficient to eliminate the symptoms of thosediseases and, perhaps, overcome the disease itself. Preferably, aneffective amount of the subject cell in the dose range of 50-10⁸ cellsfor chronic or single application and/or an effective amount of agent inthe dose range of 0.001-2.0 mg/kg yields a tissue concentration of10-10⁵ cells per ml tissue and of agent in the range of about 10⁻¹³molar to about 10⁻⁵ molar, but the concentrations may be greaterprovided that toxicity is avoided.

In the context of the present invention, the terms “treat” and “therapy”and the like refer to alleviate, slow the progression, prophylaxis,attenuation or cure of existing disease or condition that has or iscausing cell death in the CNS. “Prevent”, as used herein, refers toputting off, delaying, slowing, inhibiting, or otherwise stopping,reducing or ameliorating the onset of such diseases or disorders. It ispreferred that a large enough quantity of the cell(s) and/or agent(s) beapplied in non-toxic levels in order to provide an effective level ofactivity against the disease. The method of the present invention may beused with any animal, such as a mammal or a bird (avian), morepreferably a mammal. Poultry are a preferred bird. Exemplary mammalsinclude, but are not limited to rats, mice, cats, dogs, horses, cows,sheep, pigs, and more preferably humans.

“Therapeutic cells(s)” is defined herein to comprise at least one cellor type of cell, for example and without limitation a neural stem cell,that is transported via intranasal application to the upper third of thesubject's nasal cavity and into the damaged and/or degenerating CNS ofthe subject undergoing cell-replacement therapy. The therapeutic cell(s)may be derived from any source and may be at various stages ofdevelopmental differentiation as long as the therapeutic cell(s) aresufficient to prevent or reduce the morphological and/or behavioralneurological symptoms of the neurological disorder, disease and/orcondition being treated with cell-replacement therapy according to thepresent invention. Moreover, it is recognized that the therapeuticcell(s) may be either heterologous or autologous to the host. Byheterologous it is intended that the therapeutic cell is derived from amammal other than the patient subject, while an autologous therapeuticcell is derived from the patient subject, manipulated ex vivo, andtransported back into the patient subject's CNS by methods of thepresent invention. Therapeutic lymphocytes may also administered to theupper third of the nasal cavity using the present invention to targetboth the central nervous system and lymphatics. Lymphocytes function aspart of the body's defenses and include natural killer cells (NK cells),T cells and B cells. Such cells can be useful in the treatment of braintumors and other CNS and lymphatic disorders. Further discussion oftherapeutic cells is undertaken infra, each such aspect is included inthe definition of “therapeutic cells”.

As used herein, “regulatory agent” refers to any molecule having agrowth, proliferative, differentiative, or trophic effect on atransplanted donor cell of the present invention. Any regulatory agentthat is capable of regulating the development of the transplanted donorcell can be administered by the methods of the present invention. See,for example, Mackay-Sim et al. (2000) Prog. Neurobiol. 62:527-559,herein incorporated by reference. Further discussion of regulatoryagent(s) is undertaken infra, each such aspect is included in thedefinition of “regulatory agent”.

In the context of the present invention, the terms “treat” and “therapy”and “therapeutic” and the like refer to alleviate, slow the progression,prophylaxis, attenuation or cure of a damaged or degenerating CNSinvolving loss or death of CNS cells. The definition further comprisesputting off, delaying, slowing, inhibiting, or otherwise stopping,reducing or ameliorating the damage or degenerating CNS involving lossor death of CNS cells. The method of the present invention may be usedwith any animal, such as a mammal or a bird (avian), more preferably amammal. Poultry are a preferred bird. Exemplary mammals include, but arenot limited to rats, mice, cats, dogs, horses, cows, sheep, pigs, andmore preferably humans.

As used herein, the terms “differentiate” and “mature” refer to theprogression of a cell from a stage of having the potential todifferentiate into at least two different cellular lineages to becominga specialized cell. Such terms can be used interchangeably for thepurposes of the present invention. The term “lineage” refers to all ofthe stages of the developmental cell type, from the earliest precursorcell to a completely mature cell (i.e., a specialized cell).Accordingly, the transported therapeutic cells of the present inventioncan be derived from a multipotent cell lineage, preferably a neurallineage, and may be in any stage of differentiation. Thus, the presentinvention includes therapeutic cells that are naturally programmed todifferentiate into only one type of lineage. These types of cells caninclude some kinds of fibroblasts or simply differentiated astroglia,neurons, oligodendrocytes, microglia or endothelial cells, and they maybe derived or just isolated from the tissue of a dead donor.

Further aspects of these terms are discussed infra, each such aspect isincluded within the definition of the terms.

As used herein, the term “multipotent stem cell” refers to a cellcapable of differentiating into a variety of lineages. Multipotenttherapeutic, e.g., stem, cells are characterized by their ability toundergo continuous cellular proliferation, to regenerate exact copies ofthemselves (self-renewal), to generate a large number of regionalcellular progeny, and to elaborate new cells in response to injury ordisease. A “multipotent population of cells” refers to a composition ofcells capable of differentiating into less than all lineages of cellsbut at least into two cell lineages. Current studies have demonstratedthat multipotent stem cells from a non-neurologic region are notlineage-restricted to their developmental origin, but can generateregion-specific neurons when exposed to the appropriate environmentalcues (Lamga et al. (2001) J. Neurosci. 20:8727-8735).

A “neural stem cell” is defined herein as a multipotent cell that is animmature and uncommitted multipotent cell that exists in the nervoussystem (Ourednik et al. (1999) Clinical Genetics 56:267-278). Underspecific conditions, the neural stem cell is capable of producingdaughter cells that can terminally differentiate into neurons and glia(i.e., astrocytes (type I and II) and oligodendrocytes). They exist inboth the developing nervous system and in the adult nervous system. Adetailed characterization of the properties of neural stem cells can befound in, for example, McInnes et al. (1999) Clin. Genet. 56:267-278.

A “neuronal progenitor cell” is an undifferentiated cell that is derivedfrom a neural stem cell and which has committed to a particular path ofdifferentiation, does not exhibit self-maintenance, and underappropriate conditions will differentiate into neuroblasts (neurongenerating cells) or fibroblasts (glia generating cells). The use ofsuch multipotent neuronal cell lineages for transplantation is known inthe art. See, for example, Snyder et al.(1992) Cell 68:33, wheremultipotent neuronal cell lines have been grafted into the ratcerebellum to form neurons and glial cells. See also, Campell et al.(1995) Neuron 15:1259-1273; Fishell et al. (1995) Development121:803-812; and, Olsson et al. (1995) Eur. J. Neurosci. 10:71-85.

“Ischemia” or ischemic episode or condition is defined herein tocomprise an ischemic condition where the brain or parts of the brain donot receive enough blood flow to maintain normal neurological function,resulting in a loss or death of CNS cells and concomitant damage and/ordegeneration of the CNS. Various conditions and/or diseases can causeischemia, including but not limited to stroke. Some of the neurologicaldisorders and diseases of the CNS defined and discussed herein arecharacterized by some level of ischemia. The neurological disorders anddiseases of the CNS defined and discussed herein are amenable totreatment with the therapeutic cell replacement strategies of thepresent invention.

An “effective amount” of therapeutic cells and/or component(s) of thepharmaceutical composition of the present invention comprisingtherapeutic cells is an amount sufficient to prevent, treat, reduceand/or ameliorate the symptoms, neuronal damage and/or underlying causesof any of the referenced disorders or diseases. In some instances, an“effective amount” is sufficient to eliminate the symptoms of thosediseases and overcome the disease itself. For illustrative purposesonly, exemplary treatment regimens relating generally to the therapeuticagents disclosed herein, including dosage ranges, volumes and frequencyare provided below:

Efficacious dosage range for delivery-enhancement agents, regulatoryagents, immunosuppressive agents and/or antibiotics comprises 0.0001-1.0mg/kg.

A more preferred dosage range may be 0.005-1.0 mg/kg.

The most preferred dosage range may be 0.05-1.0 mg/kg.

The “effective amount” of therapeutic cells, i.e., efficacious dosagerange, comprises 50 cells-10⁸ cells

A more preferred dosage range for therapeutic cells comprises 10³cells-10⁸ cells.

The most preferred dosage range for therapeutic cells comprise 10⁴cells-10⁸ cells.

The dosage volume (applicable to nasal sprays or drops) range may be0.015 ml-1.0 ml.

The preferred dosage volume (applicable to nasal sprays or drops) rangemay be 0.03 ml-0.6 ml.

The brain concentrations that are likely to be achieved with the dosageranges provided above are, for a single dose: 10-10⁸ cells per ml tissueand 0.1 nM-5 μM.

Over the course of a multi-dose treatment plan, the maximum brainconcentration may be as high as 10⁶ cells per ml tissue and 50 μM fordeliver-enhancement agents, regulatory agents, immunosuppressive agentsand antibiotics.

The present invention therefore provides methods and pharmaceuticalcompositions to improve cell-based therapies used to regenerate neuraltissue that has been damaged or is undergoing degeneration by any CNSdisease or disorder, i.e., loss or death of CNS cells. CNS disordersthat are within the scope of the present invention comprise, forexample, head injury, spinal cord injury, stroke, and ischemia. CNSdisorders within the scope of the present invention also compriseneurodegenerative diseases such as, but not limited to, brain diseasesand conditions that comprise ischemia, i.e., cerebral ischemia,ischemia, stroke, neurodegeneration, neurological complications arisingfrom such as Alzheimer's disease, Parkinson's disease, Wilson's disease,Lewy body dementia, multiple sclerosis, cerebellar ataxia, progressivesupranuclear palsy, amyotrophic lateral sclerosis, affective disorders,anxiety disorders, autism and/or schizophrenia; cell damage; nervedamage from cerebrovascular disorders such as stroke in the brain orspinal cord, from CNS infections including meningitis and HIV, fromtumors of the brain and spinal cord, prion diseases, and CNS disordersresulting from ordinary aging (e.g., anosmia), brain injury, spinal cordinjury and/or metabolic disorders affecting the CNS.

Accordingly, the embodiments of the present invention find utility inenhancing the regeneration or repair of damaged neuronal tissue in ananimal having undergone a neural regenerative, i.e., cell-based,strategy that comprises the intranasal application via the upper thirdof the subject animal nasal cavity, thereby bypassing the blood-brainbarrier, of at least one therapeutic cell into the CNS of the mammal totreat a neurological disease or disorder of the CNS involving ischemiaand/or CNS cell loss or death.

Neural regenerative strategies comprising the transplantation of donorcells into the CNS of a host are known in the art. However, it is notknown to bypass the blood-brain barrier with therapeutic cells, thustransporting such cells directly into the damaged or degenerating CNS ofa host subject by intranasal application to the upper third of the nasalcavity. The therapeutic cell may be aided in transportation by at leastone delivery-enhancement agent, in viability by at least oneimmunosuppressive agent, and/or developmentally regulated by at leastone regulatory agent, while the patient may be protected from mucosalbacteria bypassing the blood-brain barrier through use of at least oneantibiotic, each of which may administered by the method of the presentinvention and, as will be further discussed below, some of thecomponents of the therapeutic method may be administered systemicallyand/or intranasally.

Transportation Pathway to Bypass Blood-Brain Barrier

The Olfactory Nerve

Various methods of the present invention include administration of thetherapeutic cells and/or pharmaceutical composition(s) of the presentinvention to tissue innervated by the olfactory nerve and that islocated in the upper third of the nasal cavity. The therapeutic cellsand/or pharmaceutical composition(s) of the present invention can bedelivered to the olfactory area via application to the upper third ofthe nasal cavity.

Fibers of the olfactory nerve are unmyelinated axons of olfactoryreceptor cells that are located in the upper one-third of the nasalmucosa. The olfactory receptor cells are bipolar neurons with swellingscovered by hair-like cilia that project into the nasal cavity. At theother end, axons from these cells collect into aggregates and enter thecranial cavity at the roof of the nose. Surrounded by a thin tube ofpia, the olfactory nerves cross the subarachnoid space containing CSFand enter the inferior aspects of the olfactory bulbs. Once thetherapeutic cells and/or pharmaceutical composition(s) of the presentinvention is applied to the upper third of nasal cavity, the therapeuticcells and/or pharmaceutical composition(s) of the present invention canundergo transport through the nasal mucosa and into the olfactory bulband other areas of the CNS, such as the anterior olfactory nucleus,frontal cortex, hippocampal formation, amygdaloid nuclei, nucleusbasalis of Meynert, hypothalamus, midbrain, cerebellum, cervical spinalcord and the like.

Neuronal Transport

Embodiments of the present method includes administration of thetherapeutic cells and/or pharmaceutical composition(s) of the presentinvention to the subject by application to the upper third of themammalian subject's nasal cavity. Application of the therapeutic cellsand/or pharmaceutical composition(s) of the present invention in thismanner ensures that the therapeutic cells and/or pharmaceuticalcomposition(s) are transported to the CNS, brain, and/or spinal cordalong a neural pathway, with reduced systemic loss and systemicexposure. A neural pathway includes transport within or along a neuron,through or by way of lymphatics running with a neuron, through or by wayof a perivascular space of a blood vessel running with a neuron orneural pathway, through or by way of an adventitia of a blood vesselrunning with a neuron or neural pathway, or through an hemangiolymphaticsystem.

The present invention comprises transportation of the therapeutic cellsand/or pharmaceutical composition(s) by way of a neural pathway, ratherthan through the circulatory system, so that regulatory agents that areunable to, or only poorly, cross the blood-brain barrier from thebloodstream into the brain can be delivered to the lymphatic system,CNS, brain, and/or spinal cord. The therapeutic cells and/orpharmaceutical composition(s) of the present invention, once past theblood-brain barrier and in the CNS, can then be delivered to variousareas of the brain or spinal cord through lymphatic channels, through aperivascular space, or transport through or along neurons. In oneembodiment, the therapeutic cells migrate to the region of damage and/ordegeneration within the CNS.

Use of a neural pathway to transport a regulatory agent to the brain,spinal cord, or other components of the central nervous system obviatesthe obstacle presented by the blood-brain barrier so that medications,i.e., therapeutic cells and/or pharmaceutical compositions of thepresent invention, that cannot normally cross that barrier, can bedelivered directly to the CNS, e.g., the brain and spinal cord. Inaddition, the present invention can provide for delivery of a moreconcentrated level of the therapeutic cells and/or pharmaceuticalcomposition(s) of the present invention to neural cells since thetherapeutic cells and/or pharmaceutical composition(s) of the presentinvention do not become diluted in fluids present in the bloodstream. Assuch, the invention provides an improved method for delivering thetherapeutic cells and/or pharmaceutical composition(s) of the presentinvention to the CNS including the brain and/or spinal cord.

The Olfactory Neural Pathway

One embodiment of the present method includes delivery of the regulatoryagent to the subject in a manner such that the regulatory agent istransported into the CNS, e.g., the brain, and/or spinal cord along anolfactory neural pathway. Typically, such an embodiment includesadministering the regulatory agent to tissue innervated by the olfactorynerve and inside the nasal cavity. The olfactory neural pathwayinnervates primarily the olfactory epithelium in the upper third of thenasal cavity, as described above. Application of the regulatory agent toa tissue innervated by the olfactory nerve can deliver the regulatoryagent to damaged neurons or cells of the CNS, brain, and/or spinal cord.Olfactory neurons innervate this tissue and can provide a directconnection to the CNS, brain, and/or spinal cord due, it is believed, totheir role in olfaction.

Delivery through the olfactory neural pathway can employ lymphatics thattravel with the olfactory nerve to the various brain areas and fromthere into dural lymphatics associated with portions of the CNS, such asthe spinal cord. Transport along the olfactory nerve can also deliverregulatory agents to an olfactory bulb. A perivascular pathway and/or ahemangiolymphatic pathway, such as lymphatic channels running within theadventitia of cerebral blood vessels, can provide an additionalmechanism for transport of therapeutic regulatory agents to the brainand spinal cord from tissue innervated by the olfactory nerve.

Therapeutic cells and/or pharmaceutical compositions thereof may beadministered to the olfactory nerve, for example, through the olfactoryepithelium located at the upper third of the nasal cavity. Suchadministration can employ extracellular or intracellular (e.g.,transneuronal) anterograde and retrograde transport of the regulatoryagent entering through the olfactory nerves to the brain and itsmeninges, to the brain stem, or to the spinal cord. Once the therapeuticcells and/or pharmaceutical composition thereof is dispensed into oronto tissue innervated by the olfactory nerve, the therapeutic cellsand/or pharmaceutical composition and/or components thereof may betransported through the tissue and travel along olfactory neurons intoareas of the CNS including the brain stem, cerebellum, spinal cord,cerebrospinal fluid, olfactory bulb, and cortical and subcorticalstructures.

The blood-brain barrier is bypassed in the present invention byapplication of the therapeutic cells and/or pharmaceuticalcomposition(s) comprising therapeutic cells by application to the upperthird of the nasal cavity. The therapeutic cells and/or pharmaceuticalcomposition of the invention migrate from the nasal mucosa throughforamina in the cribriform plate along the olfactory neural pathway andinto the CNS. See Example 1 infra providing experimental evidence thatthe blood-brain barrier is bypassed in the hypothesized manner.

Administration to the nasal cavity employing a neural pathway can thusdeliver therapeutic cells, including but not limited to eukaryotic cellsand stem cells, and/or pharmaceutical composition comprising therapeuticcells of the present invention to the lymphatic system, brain stem,cerebellum, spinal cord, and cortical and subcortical structures. Thetherapeutic cells and/or pharmaceutical composition of the presentinvention alone may facilitate this movement into the CNS, i.e., brain,and/or spinal cord. Alternatively, a carrier and/or thedelivery-enhancement agent(s) may assist in the transport of thetherapeutic cells and/or pharmaceutical composition of the presentinvention into and along the neural pathway. Administration of atherapeutic cells and/or pharmaceutical composition of the presentinvention to the upper third of the nasal cavity thus bypasses theblood-brain barrier through a transport system from the nasal mucosaand/or epithelium to the CNS, i.e., brain and spinal cord.

Various embodiments of the invention administer the therapeutic cellsand/or pharmaceutical composition(s) of the present invention to tissueinnervated by the olfactory nerves. Such nerve systems can provide adirect connection between the outside environment and the brain, thusproviding advantageous delivery of a regulatory agent to the CNS,including brain, brain stem, and/or spinal cord. The therapeutic cellsand/or pharmaceutical composition(s) of the present invention are unableto cross or inefficiently cross the blood-brain barrier from thebloodstream into the brain. Thus, the methods of the present inventionallow for the delivery of the inventive therapeutic cells and/orpharmaceutical composition(s) by way of the olfactory nerve rather thanthrough the circulatory system. This method of administration allows forthe efficient delivery of the therapeutic cells and/or pharmaceuticalcomposition(s) of the present invention to the CNS, brain, or spinalcord without systemic loss or exposure.

The immunosuppressive agent(s) and/or antibiotic(s) may be deliveredaccording to various embodiments of the present invention eithersystemically or to the upper third of the nasal cavity either alone, orin the pharmaceutical combination comprising therapeutic cell(s).

Alternative Pathways

Alternative pathways to the olfactory nerve pathway discussed abovecomprise pathways along other nerves that innervate the nasal cavity,e.g., the trigeminal pathway, well known to the skilled artisan.

Therapeutic Cells

The therapeutic cell(s) of the present invention can be derived from anyfetal or adult mammalian tissues, including bone marrow, or neuraltissues, including tissue from the hippocampus, olfactory epithelium,olfactory bulb, subventricular zone, cerebellum, spinal cord, cortex(i.e., motor or somatosensory cortex), striatum, basal forebrain(cholenergic neurons), ventral mesencephalon (cells of the substantianigra), and the locus ceruleus (neuroadrenaline cells of the centralnervous system). Moreover, the therapeutic cell(s) may include, but arenot limited to, neural and/or multipotent stem cells, neural progenitorcells, genetically engineered cells, t-cells and/or autologous cells.

The developing and the adult animal central nervous system contains apopulation of neural stem cells and progenitor cells that are ofparticular interest in the present invention as therapeutic cells.Methods of isolation and transplantation of various neural progenitorcells derived from different tissues at different developmental stagesare known in the art and include, for example, striatum cortex (Winkleret al. (1998) Mol. Cell. Neurosci. 11:99-116; Hammang et al. (1997) Exp.Neural. 147:84-95); cortex (Brustle et al. (1998) Nat. Biotechnol16:1040-1044 and Sabate et al. (1995) Nat. Genet 9:256-260); humantelencephalon (Flax et al (1998) Nature 392:18-24 and Vescovi et al,(1999) Neuron 11:951-966); hippocampus (Gage et al. (1995) J. Neurobiol.36:249-266 and Suhonen et al. (1996) Nature 383:624-627); basalforebrain (Minger et al. (1996) Exp. Neurol. 141:12-24); ventralmesencephalon (Winkler et al. (1998) Mol. Cell. Neurosci. 11:99-116;Svendsen et al. (1996) Exp. Neural 137:376-388; Hammang et al. (1997)Exp. Neurol. 147:84-95; Studer et al. (1997) Nat. Neurosci. 1:290-295;Milward et al. (1997) J. Neurosci. Res. 50:862-871); and subventricularzone (Milward et al. (1997) Milward et al. (1997) J. Neurosci. Res.50:862-871). Each of these references is herein incorporated byreference. In addition, methods for the isolation of neural stem cellprogeny and method to promote their differentiation can also be found inU.S. Pat. No. 6,071,889 and U.S. Pat. No. 6,103,530, both of which areherein incorporated by reference.

Therapeutic cells of the present invention may also be of paraneuralorigin. A preferred example of such a cell is the adrenal medullarchromafin cell. See, for example, Bjorklund et al. (1985) NeuralGrafting in the Mammalian CNS (Amsterdam: Elsevier), pp. 3-11, andLindvall et at (1997) Ann. Neurol 22: 457-468, which demonstrate theusefulness of chromafin cells for the treatment of Parkinson's disease.

Therapeutic cells of the present invention that are not of neuralorigin, but which have been altered to produce a substance ofneurological interest, are also within the inventive scope. A preferredcell type is a human foreskin fibroblast, which is easily obtained andcultured (see, for example, U.S. Pat. No. 6,060,048). Such cells arepreferably genetically altered, using methods known in the art, toexpress neuronal growth factors, neurotransmitters, neuropeptides, orenzymes involved in brain metabolism. See, for example, Gage et al.(1987) Neurosci. 23: 795-807; Rosenberg et al. (1988) Science 242:1575-1578; Shimohama et al. (1989) Mol. Brain Res. 5: 271-278; which arehereby incorporated by reference. Alternatively, therapeutic cellsderived from a non-neuronal origin, such as epidermal cells, may beconverted or transdifferentiated into different types of neuronal cells.See, for example, U.S. Pat. No. 6,087,168.

The therapeutic cell(s) of the present invention may be geneticallyaltered prior to transplantation into the host. As used herein, the term“genetically altered” refers to a cell into which a foreign nucleicacid, e.g., DNA, has been introduced. The foreign nucleic acid may beintroduced by a variety of techniques, including, but not limited to,calcium-phosphate-mediated transfection, DEAE-mediated transfection,microinjection, viral transformation, protoplast fusion, andlipofection. The genetically altered cell may express the foreignnucleic acid in either a transient or long-term manner. In general,transient expression occurs when foreign DNA does not stably integrateinto the chromosomal DNA of the transfected cell. In contrast, long-termexpression of foreign DNA occurs when the foreign DNA has been stablyintegrated into the chromosomal DNA of the transfected cell.

Such genes of interest include neurotransmitter-synthesizing enzymes(i.e., tyrosine hydrolase (TH) and cholineacetyltransferase). Suchmethods are commonly known in the art. For instance, therapeutic donorcells from various regions of the brain and at different stages ofdevelopment have been isolated and have been immortalized via geneticalteration. For example, olfactory and cerebellum cells have beenimmortalized using the viral myc (v-myc) oncogene to generate cell lineswith neuronal and glial phenotypes (Ryder et al. (1990) J. Neurobiol.21:356). Similar studies by Snyder et al. ((1992) Cell 68:33) resultedin multipotent neuronal cell lines that were engrafted into the ratcerebellum to form neurons and glial cells. In other studies, murineneuroepithelial cells were immortalized with a retrovirus vectorcontaining c-myc and were cultured with growth factors to formdifferentiated cell types similar to astrocytes and neurons (Barlett etal. (1988) Proc. Natl. Acad. Sci. USA 85:3255).

Moreover, intranasally delivered therapeutic genetically-engineeredcells of the present invention may comprise biological factories thatcan enter the CNS and release substances that are deficient or aremissing in the patients' CNS. For example, in lipid storage diseases andhereditary metabolic disorders such as phenylketoneuria (PKU), Wilson'sdisease, Tay Sachs, lysosomal storage diseases, or Nieman Pick disease,there may be an enzyme missing in the brain from birth. Therapeuticcells of the present invention may comprise that specific missingenzyme. Such genetically-engineered therapeutic cells may then bedelivered to the upper third of the nasal cavity where the cells bypassthe blood-brain barrier and enter the brain to carry out the missingmetabolic function. More generally, genetically-engineered therapeuticcells of the present invention may act as mini biological factories thatproduce and release one or more of the following: an enzyme, a growthfactor, an anti-inflammatory agent, a neurotransmitter, aneuromodulator, an anti-oxidant, etc. that can benefit the subject inneed thereof. Alternatively, therapeutic genetically-engineered cells ofthe present invention may comprise genetically-engineeredgonadotropin-releasing hormone secreting cells to increase fertility insubjects in need thereof.

Delivery-Enhancement Agents

Certain compounds, i.e., delivery-enhancement agents, may be utilized bythe present invention to assist the therapeutic cells in delivery to thecentral nervous system and the damaged regions therein. A preferreddelivery-enhancement agent comprises hyaluronidase which has beenobserved to very significantly increase delivery of therapeutic cells tothe CNS when applied to the upper third of the nasal cavity as either apretreatment administered in an effective amount prior to thetherapeutic cell application of the present invention, or as a componentof the pharmaceutical composition comprising therapeutic cells of thepresent invention, or as a separate compound administered intranasallyto the upper third of the nasal cavity substantially simultaneously asthe therapeutic cells and/or pharmaceutical composition. It is believedthat the hyaluronidase acts on hyaluronic acid in the extracellularmatrix to enhance delivery of therapeutic cells and/or pharmaceuticalcompositions comprising therapeutic cells to the CNS. Example 2 infraillustrates the increase of effectiveness by such a delivery-enhancementagent on the delivery of therapeutic cells to the CNS.

Alternative delivery-enhancement agents comprise neuregulin,migration-inducing activity and leukemia inhibitory factor. Thesedelivery-enhancement agents, e.g., hyaluronidase, lipophilic agents,neuregulin, migration-inducing activity and leukemia inhibitory factormay be used individually, or in any combination, to enhance delivery ofthe therapeutic cells to the CNS according to the present invention.Therefore, at least one delivery-enhancement agent may be used as apretreatment to transportation of the therapeutic cells and/orpharmaceutical composition and/or as a component of the pharmaceuticalcomposition comprising therapeutic cells.

Alternative delivery-enhancement agents that further enhance the mucosaldelivery of therapeutic cells and/or pharmaceutical compositioncomprising therapeutic cells of the present invention, comprise anenzyme inhibitor, particularly proteases inhibitors as is well known tothose in the art. Protease inhibitors may include, but are limited to,antipain, arphamenine A and B, benzamidine HCl, AEBSF, CA-074, calpaininhibitor I and II, calpeptin, pepstatin A, actinonin, amastatin,bestatin, boroleucine, captopril, chloroacetyl-HOLeu-Ala-Gly-NH2, DAPT,diprotin A and B, ebelactone A and B, foroxymithine, leupeptin,pepstatin A, phosphoramidon, aprotinin, puromycin, BBI, soybean trypsininhibitor, phenylmethylsulfonyl fluoride, E-64, chymostatin,1,10-phenanthroline, EDTA and EGTA.

Still further alternative delivery-enhancement agents may include, butare not limited to, surfactants, bile salts, dihydrofusidates,bioadhesive agents, phospholipid additives, mixed micelles, liposomes,or carriers, alcohols, enamines, cationic polymers, NO donor compounds,long-chain amphipathic molecules, small hydrophobic penetrationenhancers; sodium or a salicylic acid derivatives, glycerol esters ofacetoacetic acid, cyclodextrin or beta-cyclodextrin derivatives,medium-chain fatty acids, chelating agents, amino acids or saltsthereof, N-acetylamino acids or salts thereof; mucolytic agents, enzymesspecifically targeted to a selected membrane component, inhibitors offatty acid synthesis and inhibitors of cholesterol synthesis. Thepresent invention contemplates using one or more, i.e., at least one, ofthe above delivery-enhancement agents, either alone or in combinationwith the therapeutic cells as a pharmaceutical compound in an effectiveamount.

Regulatory Agents

Certain regulatory agents to regulate, inter alia, growth anddifferentiation of the delivered therapeutic cells within the CNS arewithin the scope of the present invention and include, for example, aneffective amount of regulatory agents that promote the survival of thedonor cells by modulating the immune and inflammatory response. Suchregulatory agents include, for example, cyclosporin and various otherimmunomodulators, including, interleukins (i.e., IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10); tumor necrosis factors (i.e.,TNF-alpha and TNF-beta); and, interferons (i.e., IFN-alpha, IFN-beta,IFN-gamma, IFN-omega, and IFN-tau); and any biologically active variantsthereof. Further details regarding the administration of theseimmunomodulating agents by the methods of the present invention can befound in U.S. patent Ser. No. 09/733,168, entitled “Methods forAdministering a Cytokine to the Central Nervous System and the LymphaticSystem,” filed on Dec. 9, 2000, herein incorporated by reference.

Additional regulatory agents that find use in the methods of theinvention include CAP23, a major cortical cytoskeleton-associated andcalmodulin binding protein, and GAP43, a neural growth-associatedprotein. See, for example, Frey et al. (2000) J. Cell. Biol.7:1443-1453. Further agents of interest include Osteogenic Protein-1(OP-1) which is a morphogenic protein that stimulates growth,differention, and differentiation maintenance (U.S. Pat. No. 6,153,583);sonic hedgehog, a polypeptide shown to promote the survival ofdopaminergic neurons (Miao et al. (1996) Cell Transplant 55:2-17);various other glial growth factors (U.S. Pat. Nos. 5,716,930; 6,147,190;and 5,530,109); and any biologically active variants thereof. All ofthese references are herein incorporated by reference.

Other regulatory agents of interest and within the scope of the presentinvention comprise growth factors. As used herein “growth factor” refersto a polypeptide capable of regulating the development of thetransplanted donor cell. Growth factors useful in the methods of thepresent invention include, but are not limited to, members of theneurotrophin family (i.e., nerve growth factor (NGF), brain-derivedneurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4(NT-4, also known as NT-4/5 or NT-5); fibroblast growth factors (FGFs,i.e., basic fibroblast growth factor); epidermal growth factor family(i.e., EGF, TGF.alpha., amphiregulin, heparin-binding EGF-like growthfactor (HB-EGF), batacelluin (BTC), and the neuregulin group);platelet-derived growth factor; insulin; insulin-like growth factors(i.e., IGF-I and IGF-2); ciliary neurotrophic factor (CNTF), glia cellline-derived neurotrophic factor family (GDNF) (i.e., GDNF and neurturin(NTN), persephin (PSP), and artemin (ART)); transforming growth factor.beta. superfamily (i.e., subfamilies include TGF beta 1, TGF beta 2,TGF beta 3, TGF beta 4, TGF beta 5, activin, inhibin, decapentaplegic);growth differentiation factors (GDF) (i.e., GDF1, GDF2, GDF3, GDF5,GDF6, GDF7, GDF8, GDF9, GDF9B, GDF10, GDF11, and GDF15); glia-derivednexin; activity dependent neurotrophic factor (ADNF); glial growthfactor (GGF); and the like. It is further recognized that anybiologically active variant of these growth factors is also useful inthe methods of the present invention.

The regulatory agent of present invention may be from any animal speciesincluding, but not limited to, rodent, avian, canine, bovine, porcine,equine, and, preferably, human. Preferably the regulatory agentadministered is from the same species as the animal undergoingtreatment.

Biologically active variants of regulatory polypeptides (i.e., growthfactors, such as IGF-I, NGF, and basic FGF, cytokines, etc.) are alsoencompassed by the various methods and pharmaceutical compositions ofthe present invention. Such variants should retain the biologicalactivity of the regulatory agent, particularly the ability to regulatethe development of the donor cell (i.e., promote the survival, maintainthe desired phenotype, and/or regulate the developmental cues producedby the donor cell). For example, when the regulatory polypeptide is agrowth factor, such as IGF-I, NGF-I, or a member of the FGF family, theability to bind their respective receptor sites will be retained. Suchreceptor binding activity may be measured using standard bioassays.

One such regulatory agent, a growth factor, that is useful in thepresent invention is IGF-I. The term “IGF-I” as used herein refers toinsulin-like growth factor I (IGF-I), a single-chain peptide having 70amino acids and a molecular weight of about 7,600 daltons. Insulin-likegrowth factor I stimulates mitosis and growth processes associated withcell development. The amino acid and nucleotide sequence for IGF-I isknown in the art. See, for example, U.S. Pat. No. 5,324,639 whichdiscloses the human IGF-I sequence; Genbank Accession No. X15726, whichdiscloses the sequence of bovine IGF-I; and Genbank Accession No. X06043which discloses the sequence of rat IGF-I. Each of these references isherein incorporated by reference.

In another embodiment of the present invention, the regulatory agent maycomprise a member of the FGF family of growth factors and/orbiologically active variants thereof. The fibroblast growth factorfamily encompasses a group of structurally related proteins that bindheparin with a high affinity. FGF family members have mitogen activityand induce the proliferation of a wide variety of cell types. FGF familymembers also participate in angiogenesis, differentiation, cellmigration, embryo development, and neuronal maintenance/survival. Theterm “FGF” as used herein refers to a member of the fibroblast growthfactor family including, for example, FGF-1 (acidic FGF), FGF-2 (basicFGF), FGF-3, FGF-4, FGF-5, FGF-6, FGF-8, FGF-9, FGF-98, or abiologically active fragment or variant thereof. The amino acid sequenceand methods for making many of the FGF family members are well known inthe art.

In another embodiment of the present invention, the regulatory agent maybe nerve growth factor (NGF) or a biologically active variant thereof.NGF was originally isolated as a complex having a molecular weight of130 kDa and a sedimentation coefficient of 7S. This 7S complex includedthree types of subunits, with the “.beta.” subunit carrying all of thebiological activities of NGF. Nerve growth factor stimulates mitosis andgrowth processes of cells, particularly nerve cells, and regulatesdevelopment (i.e., influences repair, survival, and differentiation).The preferred amino acid sequence for human pre-pro-NGF and human matureNGF are provided in U.S. Pat. No. 5,288,622, which is incorporatedherein by reference.

The NGF used in the present invention may be in its substantiallypurified, native, recombinantly produced form or in a chemicallysynthesized form. For example, the NGF can be isolated directly fromcells naturally expressing NGF. NGF may also be recombinantly producedin eukaryotic or prokaryotic cell expression systems as described inEdwards et al, (1988) Mol. Cell. Biol. 8:2456; U.S. Pat. No. 5,986,070;and U.S. Pat. No. 6,005,081; all of which are herein incorporated byreference. Alternatively, the regulatory agent of the present inventionmay comprise erythropoietin (EPO), brain-derived neurotrophic factor(BDNF) and epidermal growth factor (EGF). Each of the regulatory agentsdescribed herein play a crucial role in the in-vivo survival anddifferentiation of the therapeutic cells of the present inventivemethods and pharmaceutical compositions.

Administration of an effective amount of at least one regulatory agentby the methods of the present invention, i.e., intranasally to the upperthird of the nasal cavity, alone and/or in combination with thetherapeutic cells, will regulate development of the therapeutic celltransported to the CNS. The phrase “regulate development” is intendedherein to mean, inter alia, that the regulatory agent potentiates thesurvival, differentiation, axonal development, dendritic development,and/or proliferation of the transported therapeutic cell; improvesadhesion of the transported therapeutic cells to surrounding tissues(i.e., incorporation into parenchymal tissue); improves the capacity ofthe transported therapeutic cells to establish synaptic connection withthe host neurons (Le, enhances nerve fiber formation in the donor cells;increases nerve fiber projection distances of the donor cells; orenhances nerve fiber destiny of the donor cells); and/or instructs thetransported therapeutic cell to commit to a specific neural lineage(i.e., adopt a neuronal (GABA-ergic neurons, dopaminergic neurons,cholinergic neurons, hippocampal neurons, and the like), astrocytic oroligodendritic cell fate). It is further recognized that a regulatoryagent can potentiate the survival of a transplanted donor cell bymodulating the immune response of the subject. By “modulate” is intendedthe down regulation of the immune or inflammatory response (i.e.,influencing systemic immune function, antigen presentation, cytokineproduction, lymphocyte proliferation, and entry of lymphocytes andmacrophages into the CNS).

Furthermore, administration of the regulatory agent is known to“regulate development” of the invasively transplanted donor cell byinfluencing the developmental cues released by the transplanted donorcells (i.e., promote the donor cell to release neurotransmitters suchas, dopamine, acetylcholine, GABA, or other neuroprotective factors). Assuch, the function and repair (i.e., enhanced nerve fiber formation,nerve fiber projection distances, and/or nerve fiber density) of thesurrounding host tissue can be enhanced by the noninvasive methods ofthe present invention.

Delivery of an effective amount of one or more, i.e., at least one,regulatory agent to the CNS of a mammal may be achieved viaadministration of a pharmaceutical composition comprising atherapeutically effective dose of this agent. Alternatively, aneffective amount of the at least one regulatory agent may be deliveredintranasally to the upper third of the nasal cavity and/or systemicallyas a pretreatment, co-treatment and/or post-treatment to application ofthe pharmaceutical composition and/or therapeutic cell(s) of the presentinvention. By “effective amount” is meant, inter alia, the concentrationof regulatory agent that is sufficient to elicit the desired therapeuticeffect with respect to regulating the development of a donor cell, asdescribed herein. Accordingly, an effective amount of the regulatoryagent augments the clinical outcome of the cell replacement therapy incomparison to animals treated with only the cell replacement strategy.As such, a therapeutically effective dose can be assayed via a reductionin neural deficits associated with the CNS disorder being treated, andhence is characterized by an improvement in clinical symptoms.

Methods to quantify the extent of neurologic damage and to determine ifthe CNS disorder has been treated are well known to those skilled in theart. Such methods include, but are not limited to, histological methods,molecular marker assays, and functional/behavior analysis. For example,enhanced functional integration of the donor cells and/or enhancedfunction and repair of the surrounding neuronal tissue can be assayed byexamining the restoration of various functions including cognitive,sensory, motor, and endocrine. Motor tests include those that quantitaterotational movement away from the degenerative side of the brain, andthose that assay for balance, coordination, slowness of movement,rigidity, and tremors. Cognitive tests include memory tests and spatiallearning. The specific assays used to determine treatment of aneurologic disease will vary depending on the disorder.

Desired biological activities beneficial to the regulation oftransported therapeutic cell development include, for example,potentiation of the survival and/or proliferation of the transportedtherapeutic cells; improvement in the capacity of the transportedtherapeutic cell to establish synaptic connection with the host neurons;and/or instruction of the transported therapeutic cell to commit to aspecific neural lineage. Methods to assay such events are known in theart. For example, an improvement in the survival of the transportedtherapeutic cells following the administration of the regulatory agentcan be assayed using various non-invasive scans such as computerizedaxial tomography (CAT scan or CT scan), nuclear magnetic resonance ormagnet resonance imaging (NMR or MRS) or positron emission tomography(PET) scans. Alternatively, transported therapeutic cell survival can beassayed post-mortem by microscopic examination of the region oftransported therapeutic cell transplantation. The region of transportedtherapeutic cells can be identified, for example, by assaying formolecular markers specific to the transported therapeutic cells oralternatively, by prior incorporation of tracer dyes. Such dyes include,for example, rhodamine- or flourescein-labeled microspheres, fast blue,or retrovirally introduced histochemical markers.

The effective amount will depend on many factors including, for example,the CNS disorder being treated, the type of donor cell transplanted intothe mammal, and the responsiveness of the subject undergoing treatment.It is further recognized that the therapeutically effective amount willdepend on the type of developmental regulation of the transportedtherapeutic cell that is desired (i.e., potentiation of the survivaland/or proliferation of the transported therapeutic cell; improvement ofthe capacity of the transported therapeutic cell to establish synapticconnection with the host neurons; regulation of the developmental cuesreleased by the transported therapeutic cells; or improved function andrepair of the surrounding neural tissue). Methods to determine efficacyand dosage are known to those skilled in the art.

For example, in Parkinson's disease, the neurons that degenerate are thedopaminergic neurons of the substantia nigra. Cell replacementstrategies for patients with advanced Parkinson's disease are known andinclude, for example, intrastriatal grafts of nigral dopaminergicneurons from 6- to 9-week-old human embryos (Olanow at al. (1996) TrendsNeurosci. 19:102-109 and Lindvall et al. (1999) Mov. Disord.14:201-205). Delivery of pharmacologically active regulatory agents toregions of the brain affected by Parkinson's disease (i.e., midbrain andsubstantia nigra) is known in the art, however not in combination withintranasal delivery of therapeutic cells in such a way that theblood-brain barrier is bypassed.

As used herein, an “effective amount” of a regulatory agent incombination with transported therapeutic cells and/or pharmaceuticalcompositions comprising therapeutic cells of the present invention forthe treatment of Parkinson's disease using the administration method ofthe present invention will be sufficient to reduce or lessen theclinical symptoms of Parkinson's disease. As such, an effective amountof the regulatory agent (i.e., growth factor) administered by themethods of the present invention will augment the cell replacementstrategies performed under the present invention for the treatment ofParkinson's disease. Accordingly, the methods of the invention enhancesurvival and/or improve clinical status of the treated animals incomparison to animals treated with cell replacement strategy alone.Improvement in clinical status for Parkinson's disease includes, forexample, improvement in the ventral mesencephalic graft efficacy interms of apomorphine-induced rotational decrease, an increase in thedensity of striatal reinnervation, and an enhancement in neuronalsurvival (Tomqvist et al. (2000) Exp. Neurol. 164:130-138).

Huntington disease is characterized by progressive neurodegeneration,particularly in the striatum and cortex, which induces severeimpairments in both motor and cognitive functions. Current cellreplacement therapies replace inhibitor connections from the striatum toother structures such as the globus pallidus through the implantation ofstriatal precursor cells. Delivery of pharmacologically activeregulatory agents to regions of the brain that are affected byHuntington disease (i.e., caudate-putamen, thalamus, dincephalon,cerebellum, and frontal cortex) is known in the art, though never inconnection with therapeutic cells and/or pharmaceutical compositionscomprising therapeutic cells of the present invention, wherein theblood-brain barrier is bypassed.

As used herein, an “effective amount” of a regulatory agent for thetreatment of Huntington disease using the administration method of thepresent invention will be sufficient to reduce or lessen the clinicalsymptoms of Huntington disease. Thus, an effective amount of theregulatory agent (i.e., growth factor) administered by the methods ofthe present invention will augment the cell replacement strategiescommonly performed under the present invention for the treatment ofHuntington disease. As such, the methods of the invention enhancesurvival and/or improve clinical status of the treated animals incomparison to animals treated with cell replacement strategy alone.Improvement in clinical status includes, for example, disinhibition ofpallidal output, reduced locomotor hyperactivity, recovery of complexmotor and cognitive behavior, and restitution of new habit-learningsystems in the lesioned striatum. See, for example, Bjorklund et al.(1994) Functional Neural Transplantation (Raven, N.Y.), pp. 157-195;Dunnett et al. (1995) Behav. Brain Res. 66:133-142; Kendall et al.(1998) Nat. Med. 4:727-729; Palfi et al. (1998) Nat. Med. 4:963-966;Brasted et al. (1999) Proc. Natl. Acad. Sci. USA 96:10524-10529; andWictorin et al. (1992) Prog. Neurobiol. 38:611-639; all of which areherein incorporated by reference. Administration of regulatory agents bythe methods of the present invention will be sufficient to improve theclinical outcome of the cell replacement therapy. Such assays can bereadily used by one skilled in the art to determine the dosage rangeand/or appropriate regulatory agent of choice for the effectivetreatment of Huntington disease.

Ischemic damage to the CNS (and resulting cell loss and death) canresult from, for example, cardiac arrest or coronary artery occlusion,or cerebral artery occlusion or stroke. Neural circuits of the CNSdamaged following an ischemic event have been reconstructed usingvarious cell replacement strategies. For instance, for focal ischemiaevents, implantation of embryonic striatum into the damaged striatum(Hodges et al. (1994) Functional Neural Transplantation (Raven, N.Y.),pp. 347-386) and implantation of neurons derived from a humanteratocarcinoma cell line (Borlongan et al. (1998) Exp. Neurol.149:310-321 and Borlongan et al. (1998) Neuroreport 9:3703-3709) havebeen performed. See also, for example, Hodges et al. (1996) Neurosci.72:959-988, Sorensen et al. (1996) Exp. Neurol. 138:227-235, and Sindenet al. (1997) Neurosci. 81:599-608.

As used herein, an “effective amount” of a regulatory agent for thetreatment of ischemic injury will be sufficient to reduce or lessen theclinical symptoms of the ischemic event. As such, an effective amount ofthe regulatory agent administered by the methods of the presentinvention will augment the cell replacement strategies commonlyperformed according to the present invention for the treatment of anischemic injury. Improvement in clinical status includes, for example, areduction in infarct size, edema, and/or neurologic deficits (Le.,improved recovery of motor, sensory, vestibulomotor, and/orsomatosensory function). Improvements further encompass a reduction inneural deficits, and hence improved recovery of motor, sensory,vestibulomotor, and/or somatosensory function.

Methods to determine if an ischemic event has been treated, particularlywith regard to reduction of ischemic damage including infarct size,edema, and development of neural deficits, are well known to thoseskilled in the art. For example, after ischemic injury, there is asignificant increase in the density of omega 3 (peripheral-typebenzodiazepine) binding sites (Benazodes et al. (1990) Brain Res.522:275-289). Methods to detect omega 3 sites are known and can be usedto determine the extent of ischemic damage. See for example, Gotti etal. (1990) Brain Res. 522:290-307 and references cited therein.Alternatively, Growth Associated Protein-43 (GAP-43) can be used as amarker for new axonal growth following an ischemic event. See, forexample, Stroemer et al. (1995) Stroke 26:2135-2144, and Vaudano et al.(1995) J. Neurosci 15:3594-3611. The therapeutic effect may also bemeasured by improved motor skills, cognitive function, sensoryperception, speech and/or a decrease in the propensity to seizure in themammal undergoing treatment. Such functional/behavior tests used toassess sensorimotor and reflex function are described in, for example,Bederson et al. (1986) Stroke 17:472-476, DeRyck et al. (1992) BrainRes. 573:44-60, Markgraf et al. (1992) Brain Res. 575:238-246, Alexis etal. (1995) Stroke 26:2338-2346. Enhancement of neuronal survival mayalso be measured using the Scandinavian Stroke Scale (SSS) or theBarthel Index. Such assays can be readily used by one skilled in the artto determine the dosage range and/or appropriate regulatory agent ofchoice for the effective treatment of an ischemic event.

For purposes of regulating the development of a therapeutic cell(s) ofthe present invention following intranasal transportation into the CNSwith bypass of the blood-brain barrier in a mammal, the therapeuticallyeffective amount or dose of a regulatory agent may comprise about 0.002mg/kg to about 2.0 mg/kg of body weight or from about 0.03 mg/kg toabout 0.6 mg/kg of body weight. Alternatively, the regulatory agent maybe administered at 0.0004, 0.001, 0.005, 0.007, 0.009, 0.01, 0.04, 0.06,0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, or 2.0 mg/kg ofbody weight. It is further recognized that a lower dose range of certainregulatory agents (i.e., ADNF) may be preferred. In these embodiments,the regulatory agent can be administered from about 0.1 ng/kg to about20 ng/kg. Alternatively, the regulatory agent can be administered at0.2, 0.4, 0.6, 0.8, 1, 2, 4, 8, 12, 15, 18, and 19 ng/kg of body weight.

Antibiotic Agents

In various embodiments, the present invention may further comprise aneffective amount of at least one antibiotic, or alternatively at leastone antibiotic(s) pretreatment administered prior to application of thepharmaceutical composition to the upper third of the nasal cavity may beused, or any combination thereof, to protect the patient undergoingtherapeutic cell therapy. Further, the antibiotic(s) may be delivered asa pretreatment, co-treatment and/or post treatment systemically and/orby application to the upper third of the nasal cavity. The utility ofsuch an antibiotic element within the present invention is to reduce therisk that bacteria found in the nasal cavity may enter the nasal tissuesat the upper third of the nasal cavity during application of thetherapeutic cell(s) and/or pharmaceutical composition, cross theblood-brain barrier and infect other tissues within the CNS. Particulartissues of concern include, but are not limited to, the brain, meninges,blood, spinal cord, and other peripheral tissues. A preferred embodimentis to pretreat and/or simultaneously treat the patient withantibiotic(s) when the delivery-enhancement agent(s), e.g.,hyaluronidase, is applied to the upper third of the nasal cavity.

Exemplary antibiotics for use in the present invention comprisemupirocin, defensin, gentamycin, geneticin, cefminoxime, penicillin,streptomycin, xylitol, or other antibiotic, either alone or incombination to assist in protecting the patient who is receivingtherapeutic cell(s) and/or pharmaceutical composition of the presentinvention. The use of such antibiotics within nasal treatments is widelyreported in the literature as will be readily recognized by the skilledartisan, however no such nasal treatment is reported in conjunction withthe intranasal application of therapeutic cells and/or pharmaceuticalcompositions comprising therapeutic cells to the upper third of thenasal cavity whereby the blood-brain barrier is bypassed.

Immunosuppressive Agents

Alternate embodiments of the present invention may further comprise aneffective amount of at least one immunosuppressive agent to enhance theviability of the therapeutic cell(s) through protection frominflammatory response and/or activation of host immunocompetent cells.The immunosuppressive agent(s) may be delivered either as apretreatment, simultaneously with the therapeutic cell(s) and/orpharmaceutical composition and/or post-treatment of the therapeuticcell(s) and/or pharmaceutical composition. Such immunosuppressivetherapy in combination with the therapeutic cell(s) and/orpharmaceutical composition applied to the upper third of the nasalcavity will improve the survival of such cells.

When the host immunocompetent cells of the CNS, nasal mucosa and theneural pathway between the nasal mucosa and the CNS detect the appliedtherapeutic cells of the present invention, inflammatory response and/oractivation of host immunocompetent cells may result. This series ofevents will decrease the therapeutic cell(s) survival. Therefore,immunosuppression agent(s) may be employed, prior to, during and/orafter the application of therapeutic cell(s) to the upper third of thenasal cavity to play a crucial role in the survival and viability of thetherapeutic cells, The immunosuppression agent(s) may be appliedintranasally to the upper third of the nasal cavity and/or systemically.Conventional and well known immunosuppressive agents that may be usedalone, or in combination, in the present invention comprise cyclosporineA, tacrolimus, prednisolone, azathioprine, methylprednisolone,mycophenylate mophetil and sirolimus. Another immunosuppressive agentcomprises application of genetically engineered cells expressing the Fasligand.

Pharmaceutical Composition

In addition to the effective amount of at least one therapeutic celladministered to the upper third of the mammalian nasal cavity, apharmaceutical composition may be applied or administered to the upperthird of the nasal cavity. Such a pharmaceutical composition maycomprise, in addition to the effective amount of at least onetherapeutic cell, for example, the composition can comprise at least oneregulatory agent as described supra, at least one delivery-enhancementagent as described supra, at least one antibiotic, and/or at least oneimmunosuppressive agent, all as described supra and as will be discussedfurther infra. The pharmaceutical composition of the present inventionmay be combined with pre-, co-, and post-treatment with any combinationof systemic and/or application to the upper third of the nasal cavity ofthe at least one regulatory agent, delivery-enhancement agent,antibiotic and/or immunosuppressive agent.

Among the alternatives that may be combined with therapeutic cells inthe pharmaceutical composition are delivery-enhancement agents, such aslipophilic agents, that can enhance absorption of the regulatory agentthrough the mucosa or epithelium of the nasal cavity to reach damagedand/or degenerating cells in the CNS. The regulatory agent may be mixedwith a lipophilic agent or adjuvant alone or in combination with acarrier, or may be combined with one or several types of micelle orliposome substances. Among the preferred lipophilic substances arecationic liposomes including one or more of phosphatidyl choline,lipofectin, DOTAP, or the like.

A preferred delivery-enhancement agent comprises hyaluronidase which hasbeen observed to very significantly increase delivery of therapeuticcells to the CNS when applied to the upper third of the nasal cavity aseither a pretreatment to the therapeutic cell application of the presentinvention, or as a component of the pharmaceutical compositioncomprising therapeutic cells of the present invention. Alternativedelivery-enhancement agents comprise neuregulin and migration-inducingactivity. These delivery-enhancement agents, e.g., hyaluronidase,lipophilic agents, neuregulin and migration-inducing activity may beused individually, or in any combination, to enhance delivery of thetherapeutic cells to the CNS according to the present invention.Therefore, at least one delivery-enhancement agent may be used as apretreatment to transportation of the therapeutic cells and/orpharmaceutical composition and/or as a component of the pharmaceuticalcomposition comprising therapeutic cells.

The pharmaceutical composition of the present invention may furthercomprise at least one antibiotic, or alternatively an antibioticpretreatment prior to application of the pharmaceutical composition tothe upper third of the nasal cavity may be used, or any combinationthereof, to protect the patient undergoing therapeutic cell therapy.Further, the antibiotic may be delivered as a pretreatment, co-treatmentand/or post treatment given intranasally and/or systemically. Theutility of such an antibiotic element within the present invention is toreduce the risk that bacteria found in the nasal cavity may enter thenasal tissues at the upper third of the nasal cavity during applicationof the therapeutic cell(s) and/or pharmaceutical composition, cross theblood-brain barrier and infect other tissues within the CNS. Particulartissues of concern include, but are not limited to, the brain, meninges,blood, spinal cord, and other peripheral tissues. A preferred embodimentis to pretreat and/or simultaneously treat the patient with antibioticwhen a delivery-enhancement agent such as hyaluronidase is applied,either alone or in a pharmaceutical composition, to the upper third ofthe nasal cavity.

Exemplary antibiotics for use in the present invention comprisemupirocin, defensin, gentamycin, geneticin, cefminoxime, penicillin,streptomycin, xylitol, or other antibiotic, either alone or incombination to assist in protecting the patient who is receivingtherapeutic cell(s) and/or pharmaceutical composition of the presentinvention. The use of such antibiotics within nasal treatments is widelyreported in the literature as will be readily recognized by the skilledartisan.

The present invention may further comprise at least oneimmunosuppressive agent, delivered either as a pretreatment,simultaneously with the therapeutic cell(s) and/or pharmaceuticalcomposition and/or post-treatment of the therapeutic cell(s) and/orpharmaceutical composition. Such immunosuppressive therapy incombination with the therapeutic cell(s) and/or pharmaceuticalcomposition applied to the upper third of the nasal cavity will improvethe survival of such cells. When the host immunocompetent cells of theCNS, nasal mucosa and the neural pathway between the nasal mucosa andthe CNS detect the applied therapeutic cells of the present invention,inflammatory response and/or activation of host immunocompetent cellsmay result. This series of events will decrease the therapeutic cell(s)survival. Therefore, immunosuppression agent(s) may be employed, priorto, during and/or after the application of therapeutic cell(s) to theupper third of the nasal cavity to play a crucial role in the survivaland viability of the therapeutic cells. The immunosuppression agent(s)may be applied intranasally to the upper third of the nasal cavityand/or systemically. Conventional and well known immunosuppressiveagents that may be used alone, or in combination, in the presentinvention comprise cyclosporine A, tacrolimus, prednisolone,azathioprine, methylprednisolone, mycophenylate mophetil and sirolimus.Another immunosuppressive agent comprises application of geneticallyengineered cells expressing the Fas ligand.

Further, the pharmaceutical composition of the present invention maycomprise any pharmaceutically acceptable additive, carrier, and/oradjuvant that can promote the transfer of this agent within or through atissue innervated by the trigeminal nerve or olfactory nerve or along orthrough a neural pathway.

By “pharmaceutically acceptable carrier” is intended a carrier that isconventionally used in the art to facilitate the storage,administration, and/or the biological activity of therapeutic cell(s),regulatory agent(s), delivery-enhancement agent(s), antibiotic(s) and/orimmunosuppressive agent within a pharmaceutical composition of thepresent invention. A carrier may also reduce any undesirable sideeffects of the components of such a pharmaceutical composition. Asuitable carrier should be stable, i.e., incapable of reacting withother ingredients in the formulation. It should not produce significantlocal or systemic adverse effect in recipients at the dosages andconcentrations employed for treatment. Such carriers are generally knownin the art.

Suitable carriers for the various embodiments of the present inventioninclude those conventionally used for large stable macromolecules suchas albumin, gelatin, collagen, polysaccharide, monosaccharides,polyvinylpyrrolidone, polylactic acid, polyglycolic acid, polymericamino acids, fixed oils, ethyl oleate, liposomes, glucose, sucrose,lactose, mannose, dextrose, dextran, cellulose, mannitol, sorbitol,polyethylene glycol (PEG), and the like. A further pharmaceuticalcomposition may comprise microparticles, organic and inorganic compoundsserving as an adherence material for the cell(s) and cell conglomeratesthat may be transported to the CNS in various embodiments of the presentinvention, thus diminishing the loss of cells transported from the nasalmucosa to the CNS. These compounds may include several kinds of adhesivemolecules, gels (serving as an encapsulating/embedding material for thecells), components of extracellular matrix or matrices, and organicand/or inorganic particles such as fibrin or fibronectin carbon- orclay- and dextran particles and their composition.

Water, saline, aqueous dextrose, and glycols are preferred liquidcarriers, particularly (when isotonic) for solutions. The carrier can beselected from various oils, including those of petroleum, animal,vegetable or synthetic origin, for example, peanut oil, soybean oil,mineral oil, sesame oil, and the like. Suitable pharmaceuticalexcipients include starch, cellulose, talc, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate,sodium stearate, glycerol monostearate, sodium chloride, dried skimmilk, glycerol, propylene glycol, water, ethanol, and the like. Thecompositions can be subjected to conventional pharmaceutical expedients,such as sterilization, and can contain conventional pharmaceuticaladditives, such as preservatives, stabilizing agents, wetting, oremulsifying agents, salts for adjusting osmotic pressure, buffers, andthe like. Where the carrier is a liquid, it is preferred that thecarrier be hypotonic or isotonic with body fluids and have a pH withinthe range of 4.5-8.5.

Other acceptable components in the pharmaceutical composition comprise,without limitation, isotonicity-modifying agents such as water, saline,and buffers including phosphate, citrate, succinate, acetic acid, andother organic acids or their salts. Typically, the pharmaceuticallyacceptable carrier also includes one or more stabilizers, reducingagents, anti-oxidants and/or anti-oxidant chelating agents. The use ofbuffers, stabilizers, reducing agents, anti-oxidants and chelatingagents in the preparation of protein-based compositions, particularlypharmaceutical compositions, is well known in the art. See, Wang et al.(1980) J. Parent. Drug Assn, 34(6):452-462; Wang et al. (1988) J.Parent. Sci. Tech. 42:S4-S26 (Supplement); Lachman et al. (1968) Drugand Cosmetic Industry 102(1):36-38, 40, and 146-148; Akers (1988) J.Parent. Sci. Tech. 36(5):222-228; and Methods in Enzymology, Vol. XXV,ed. Colowick and Kaplan, “Reduction of Disulfide Bonds in Proteins withDithiothreitol,” by Konigsberg, pp. 185-188.

Various embodiments of the pharmaceutical composition of the presentinvention comprise suitable buffers such as acetate, adipate, benzoate,citrate, lactate, maleate, phosphate, tartarate, borate,tri(hydroxymethyl aminomethane), succinate, glycine, histidine, thesalts of various amino acids, or the like, or combinations thereof. SeeWang (1980) supra at page 455. Suitable salts and isotonicifiers includesodium chloride, dextrose, mannitol, sucrose, trehalose, or the like.

Various embodiments of the pharmaceutical composition of the presentinvention may further comprise suitable reducing agents, which maintainthe reduction of reduced cysteines, include dithiothreitol (DTT alsoknown as Cleland's reagent) or dithioerythritol at 0.01% to 0.1% wt/wt;acetylcysteine or cysteine at 0.1% to 0.5% (pH 2-3); and thioglycerol at0.1% to 0.5% (pH 3.5 to 7.0) and glutathione. Suitable antioxidantsinclude sodium bisulfite, sodium sulfite, sodium metabisulfite, sodiumthiosulfate, sodium formaldehyde sulfoxylate, and ascorbic acid.Suitable chelating agents, which chelate trace metals to prevent thetrace metal catalyzed oxidation of reduced cysteines, include citrate,tartarate, ethylenediaminetetraacetic acid (EDTA) in its disodium,tetrasodium, and calcium disodium salts, and diethylenetriaminepentaacetic acid (DTPA). See, e.g., Wang (1980) supra at pages 457-458and 460-461, and Akers (1988) supra at pages 224-227.

Various embodiments of the pharmaceutical composition of the presentinvention may further comprise one or more preservatives such as phenol,cresol, paraaminobenzoic acid, BDSA, sorbitrate, chlorhexidine,benzalkonium chloride, or the like. Suitable stabilizers includecarbohydrates such as trehalose or glycerol. The composition can includea stabilizer such as one or more of microcrystalline cellulose,magnesium stearate, mannitol, or sucrose to stabilize, for example, thephysical form of the composition; and one or more of glycine, arginine,hydrolyzed collagen, or protease inhibitors to stabilize, for example,the chemical structure of the composition.

Various embodiments of the pharmaceutical composition of the presentinvention may also comprise suitable suspending agents such ascarboxymethyl cellulose, hydroxypropyl methylcellulose, hyaluronic acid,alginate, chondroitin sulfate, dextran, maltodextrin, dextran sulfate,or the like. The composition can include an emulsifier such aspolysorbate 20, polysorbate 80, pluronic, triolein, soybean oil,lecithins, squalene and squalanes, sorbitan treioleate, or the like.

The pharmaceutical composition of the present invention may furthercomprise at least one antimicrobial such as phenylethyl alcohol, phenol,cresol, benzalkonium chloride, phenoxyethanol, chlorhexidine,thimerosol, or the like. Suitable thickeners include naturalpolysaccharides such as mannans, arabinans, alginate, hyaluronic acid,dextrose, or the like; and synthetic ones like the PEG hydrogels of lowmolecular weight; and aforementioned suspending agents may be includedin the pharmaceutical composition of the present invention.

The inventive pharmaceutical composition may further comprise include anadjuvant such as cetyl trimethyl ammonium bromide, BDSA, cholate,deoxycholate, polysorbate 20 and 80, fusidic acid, or the like. Suitablesugars include glycerol, threose, glucose, galactose, mannitol, andsorbitol.

Various embodiments of the pharmaceutical composition of the presentinvention may further comprise one or more of a solubility enhancingadditive, preferably a cyclodextrin; a hydrophilic additive, preferablya monosaccharide or oligosaccharide; an absorption promoting additive,preferably a cholate, a deoxycholate, a fusidic acid, or a chitosan; acationic surfactant, preferably a cetyl trimethyl ammonium bromide; aviscosity enhancing additive, preferably to promote residence time ofthe composition at the site of administration, preferably acarboxymethyl cellulose, a maltodextrin, an alginic acid, a hyaluronicacid, or a chondroitin sulfate; or a sustained release matrix,preferably a polyanhydride, a polyorthoester, a hydrogel, a particulateslow release depo system, preferably a polylactide co-glycolides (PLG),a depo foam, a starch microsphere, or a cellulose derived buccal system;a lipid-based carrier, preferably an emulsion, a liposome, a niosome, ora micelle. The composition can include a bilayer destabilizing additive,preferably a phosphatidyl ethanolamine; a fusogenic additive, preferablya cholesterol hemisuccinate.

The pharmaceutical composition may additionally include a solubilizingcompound to enhance stability of the regulatory agent or biologicallyactive variant thereof. For IGF-I, a preferred solubilizing agentincludes a guanidinium group that is capable of enhancing itssolubility. Examples of such solubilizing compounds include the aminoacid arginine, as well as amino acid analogs of arginine that retain theability to enhance solubility of IGF-I or biologically active variantthereof at pH 5.5 or greater. Such analogs include, without limitation,dipeptides and tripeptides that contain arginine. By “enhancing thesolubility” is intended increasing the amount of growth factor orbiologically active variant thereof that can be dissolved in solution atpH 5.5 or greater in the presence of a guanidinium-containing compoundcompared to the amount of this protein that can be dissolved at pH 5.5or greater in a solution with the same components but lacking theguanidinium-containing compound. The ability of a guanidinium-containingcompound to enhance the solubility of the growth factor or biologicallyactive variant thereof can be determined using methods well known in theart. In general, it is known to provide the concentration of thesolubilizing compound present in the composition in the range from about10 mM to about 1 M, and, for example, in the case of the compoundarginine, in a concentration range of about 20 mM to about 200 mM.

These lists of carriers and additives are by no means complete, and aworker skilled in the art can choose excipients from the GRAS (generallyregarded as safe) list of chemicals allowed in the pharmaceuticalpreparations and those that are currently allowed in topical andparenteral formulations.

Moreover, the method for formulating a pharmaceutical composition isgenerally known in the art. A thorough discussion of formulation andselection of pharmaceutically acceptable carriers, stabilizers, andisomolytes can be found in Remington's Pharmaceutical Sciences(18.sup.th ed.; Mack Publishing Company, Eaton, Pa., 1990), hereinincorporated by reference.

For the purposes of this invention, the pharmaceutical composition asdescribed herein can be formulated in a unit dosage and in a form suchas a solution, suspension, or emulsion for application to the upperthird of the nasal cavity. The pharmaceutical composition to be appliedand administered to the upper third of the nasal cavity to the tissueinnervated by the olfactory neurons may be in the form of a powder, agranule, a solution, a spray (e.g., an aerosol), an ointment, aninfusion, a drop, or a sustained-release composition, such as a polymerdisk. Other forms of compositions for administration include asuspension of a particulate, such as an emulsion, a liposome, an insertthat releases the pharmaceutical composition slowly, and the like. Thepowder or granular forms of the pharmaceutical composition may becombined with a solution and with a diluting, dispersing, or surfaceactive regulatory agent. The composition can also be in the form oflyophilized powder, which can be converted into solution, suspension, oremulsion before administration. The pharmaceutical compositioncomprising at least one regulatory agent is preferably sterilized bymembrane filtration and is stored in unit-dose or multi-dose containerssuch as sealed vials or ampoules.

Administration of Therapeutic Cells and/or Pharmaceutical Compounds

Administering therapeutic cells according to the methods of theinvention may include application of therapeutic cells alone orformulating the therapeutic cells with one or more of the compoundsdescribed supra as pharmaceutical compositions and administering thepharmaceutical compositions to an animal subject or host, including ahuman patient, intranasally to the upper third of the nasal cavity. Thetherapeutic cells and/other components of the pharmaceutical compositionthereof, e.g., delivery-enhancement agent, regulatory agent, antibioticand/or immunosuppressive agent, may be administered at one of a varietyof doses sufficient to provide an effective amount at the desired pointof action of the therapeutic cell and/or pharmaceutical compositioncomponent. Doses for humans and other mammals can range from about 0.001mg/kg to about 100 mg/kg, preferably from about 0.01 mg/kg to about 10mg/kg, preferably from about 0.1 mg/kg to about 1-10 mg/kg. As noted,delivery-enhancement agent(s), regulatory agent(s), antibiotic(s) and/orimmunosuppressive agent(s) may be delivered as pre-treatment,co-treatment and/or post-treatment with the therapeutic cell(s) and/orpharmaceutical composition, either alone or as a component of thepharmaceutical composition, and, when not comprised within thepharmaceutical composition, may be delivered either systemically or tothe upper third of the nasal cavity.

For application to the upper third of the nasal cavity as suspensions,aerosols, sprays or drops, the therapeutic cell(s) and/or pharmaceuticalcomposition(s) can be prepared according to techniques well known in theart of pharmaceutical formulation. The compositions can be prepared assuspensions of cells in solutions which may comprise salts such assaline, components such as phosphate, succinate or citrate buffers tomaintain pH, osmoregulatory and osmotic agents such as taurine, andsuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons or other solubilizing or dispersing agents known in theart. The means of applying a pharmaceutical composition intranasally tothe upper third of the nasal cavity may be in a variety of forms such asa powder, spray, gel or nose drops.

Other forms of compositions for administration of therapeutic cellsand/or pharmaceutical compositions or elements thereof include asuspension of a particulate, such as an emulsion, a liposome, or in asustained-release form to prolong the presence of the pharmaceuticallyactive agent in an individual. The powder or granular forms of thepharmaceutical composition may be combined with a solution and with adiluting, dispersing or surface-active agent. Additional compositionsfor administration include a bioadhesive to retain the agent at the siteof administration at the upper third of the nasal cavity, for example aspray, paint, or swab applied to the mucosa. A bioadhesive can refer tohydrophilic polymers, natural or synthetic, which, by the hydrophilicdesignation, can be either water soluble or swellable and which arecompatible with the pharmaceutical composition. Such adhesives functionfor adhering the formulations to the mucosal tissues of the upper thirdof the nasal cavity. Such adhesives can include, but are not limited to,hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, ethylcellulose, carboxymethyl cellulose, dextran, guargum, polyvinyl pyrrolidone, pectins, starches, gelatin, casein, acrylicacid polymers, polymers of acrylic acid esters, acrylic acid copolymers,vinyl polymers, vinyl copolymers, polymers of vinyl alcohols, alkoxypolymers, polyethylene oxide polymers, polyethers, and combinationsthereof. The composition can also be in the form of lyophilized powder,which can be converted into solution, suspension, or emulsion beforeadministration. The pharmaceutical composition is preferably sterilizedby membrane filtration and is stored in unit-dose or multi-dosecontainers such as sealed vials or ampoules.

The pharmaceutical composition may be formulated in a sustained-releaseform to prolong the presence of the active agent in the treatedindividual. Many methods of preparation of a sustained-releaseformulation are known in the art and are disclosed in Remington'sPharmaceutical Sciences. Generally, the therapeutic cells,pharmaceutical composition and/or components of the pharmaceuticalcomposition, i.e., delivery-enhancement agent, regulatory agent,antibiotic and/or immunosuppressive agent, may be entrapped insemi-permeable matrices of solid hydrophobic polymers. The matrices canbe shaped into films or microcapsules. Matrices can include, but are notlimited to, polyesters, co-polymers of L-glutamic acid and gammaethyl-L-glutamate, polylactides, polylactate polyglycolate, hydrogels,non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolicacid copolymers, hyaluronic acid gels, and alginic acid suspensions.Suitable microcapsules can also include hydroxymethylcellulose orgelatin and poly-methyl methacrylate. Microemulsions or colloidal drugdelivery systems such as liposomes and albumin microspheres can also beused.

Delivery Systems

Therapeutic cells and/or a pharmaceutical composition comprisingtherapeutic cells and/or components of the pharmaceutical composition ofthe present invention may further be dispensed and applied intranasallyto the upper third of the nasal cavity as a powdered or liquid nasalspray, suspension, nose drops, a gel, film or ointment, through a tubeor catheter, by syringe, by packtail, by pledget (a small flat absorbentpad), by nasal tampon or by submucosal infusion. In some aspects of thepresent invention, the methods comprise administering to an individualtherapeutic cells and/or a pharmaceutical composition thereof to theupper third of the nasal cavity by way of a delivery device. Nasal drugdelivery can be carried out using devices including, but not limited to,unit dose containers, pump sprays, droppers, squeeze bottles, airlessand preservative-free sprays, nebulizers (devices used to change liquidmedication to an aerosol particulate form), metered dose inhalers, andpressurized metered dose inhalers. In some aspects, an accurateeffective dosage amount is contained within a bioadhesive patch that isplaced directly within and on the upper third of a nasal cavity.

Therapeutic cells and/or a pharmaceutical composition comprisingtherapeutic cells and/or components of the therapeutic composition ofthe present invention may be conveniently delivered to the upper thirdof the nasal cavity in the form of an aerosol spray using a pressurizedpack or a nebulizer and a suitable propellant including, but not limitedto, dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, hydrocarbons, compressed air, nitrogen orcarbon dioxide. An aerosol system requires the propellant to be inerttowards the therapeutic cells and/or pharmaceutical composition as willbe readily recognized by the skilled artisan. In the case of apressurized aerosol, the dosage unit may be controlled by providing avalve to deliver an accurately metered amount.

The means to deliver therapeutic cells or pharmaceutical compositioncomprising therapeutic cells and/or components of the pharmaceuticalcomposition of the present invention to the upper third of the nasalcavity as a powder may be in a form such as microspheres delivered by anasal insufflator device (a device to blow a gas, powder, or vapor intoa cavity of the body) or pressurized aerosol canister. The insufflatorproduces a finely divided cloud of the dry powder or microspheres. Theinsufflator may be provided with means to ensure administration of asubstantially metered amount of the pharmaceutical composition. Thepowder or microspheres should be administered in a dry, air-dispensableform. The powder or microspheres may be used directly with aninsufflator which is provided with a bottle or container for the powderor microspheres. Alternatively the powder or microspheres may be filledinto a capsule such as a gelatin capsule, or other single dose deviceadapted for nasal administration. The insufflator can have means such asa needle to break open the capsule or other device to provide holesthrough which jets of the powdery composition can be delivered to theupper third of the nasal cavity. In this embodiment, the therapeuticcells may be dehydrated and/or lyophilized, with subsequent rehydrationin the nasal mucosa.

Intermittent and Cyclic Dosing

In various embodiments of the invention, therapeutic cells and/or apharmaceutical composition comprising an effective amount of thetherapeutic cells and/or the components of the pharmaceuticalcomposition may be administered as a single and one-time dose, oralternatively therapeutic cells and/or the components of thepharmaceutical composition may be administered more than once andintermittently. By “intermittent administration” is intendedadministration of an effective amount of therapeutic cells and/or thecomponents of the pharmaceutical composition, followed by a time periodof discontinuance, which is then followed by another administration ofan effective amount, and so forth. Administration of the effectiveamount of therapeutic cells and/or the components of the pharmaceuticalcomposition may be achieved in a continuous manner, as for example witha sustained-release formulation, or it may be achieved according to adesired daily dosage regimen, as for example with one, two, three, ormore administrations per day. By “time period of discontinuance” isintended a discontinuing of the continuous sustained-released or dailyadministration of the therapeutic cells and/or the components of thepharmaceutical composition. The time period of discontinuance may belonger or shorter than the period of continuous sustained-release ordaily administration. During the time period of discontinuance, thetherapeutic cells and/or the components of the pharmaceuticalcomposition level in the relevant tissue is substantially below themaximum level obtained during the treatment. The preferred length of thediscontinuance period depends on the concentration of the effective doseand the form of therapeutic cells and/or the components of thepharmaceutical composition used. The discontinuance period can be atleast 2 days, preferably is at least 4 days, more preferably is at least1 week and generally does not exceed a period of 4 weeks. When asustained-release formulation is used, the discontinuance period must beextended to account for the greater residence time of regulatory agentat the site of injury. Alternatively, the frequency of administration ofthe effective dose of the sustained-release formulation can be decreasedaccordingly. An intermittent schedule of administration of therapeuticcells and/or the components of the pharmaceutical composition maycontinue until the desired therapeutic effect, and ultimately treatmentof the disease or disorder is achieved.

In yet another embodiment, intermittent administration of the effectiveamount(s) of therapeutic cells and/or the components of thepharmaceutical composition is cyclic. By “cyclic” is intendedintermittent administration accompanied by breaks in the administration,with cycles ranging from about 1 month to about 2, 3, 4, 5, or 6 months.For example, the administration schedule might be intermittentadministration of the effective dose of therapeutic cells and/or thecomponents of the pharmaceutical composition, wherein a singleshort-term dose is given once per week for 4 weeks, followed by a breakin intermittent administration for a period of 3 months, followed byintermittent administration by administration of a single short-termdose given once per week for 4 weeks, followed by a break inintermittent administration for a period of 3 months, and so forth. Asanother example, a single short-term dose may be given once per week for2 weeks, followed by a break in intermittent administration for a periodof 1 month, followed by a single short-term dose given once per week for2 weeks, followed by a break in intermittent administration for a periodof 1 month, and so forth. A cyclic intermittent schedule ofadministration of therapeutic cells and/or the components of thepharmaceutical composition to a subject may continue until the desiredtherapeutic effect, and ultimately treatment of the disorder or diseaseis achieved.

For purposes of regulating therapeutic cell development and therebyreducing or preventing the clinical manifestation of the neurologicaldisorder being treated, intranasal administration of one or moretherapeutically effective doses of at least one regulatory agent mayoccur within minutes, hours, days, or even weeks of the initialapplication of the therapeutic cells and/or pharmaceuticalcomposition(s) of the present invention. For example, the initialtherapeutic dose of the at least one regulatory agent may beadministered within about 2 to 4 hours, within about 2 to 6 hours,within about 8 hours, within about 10 hours, about 15 hours, about 24hours, within about 36 hours, 48 hours, 72 hours, or about 96 hours, orlonger following application of the therapeutic cells and/orpharmaceutical composition(s) of the present invention. One or moreadditional doses of the regulatory agent may be administered for hours,days, or weeks following the initial dose. Furthermore, the animalundergoing a cell replacement regeneration therapy according toembodiments of the present invention may be administered additionalregulatory agents and/or therapeutic cells and/or pharmaceuticalcompositions intermittently over time according to a patient carestrategy. Thus, for example, an animal undergoing cell replacementtherapy can be administered one or more therapeutically effective dosesof the regulatory agent(s), therapeutic cells and/or pharmaceuticalcomposition(s) of the present invention thereof prior to, during, orfollowing the initial application. Similarly, the delivery enhancementagent, immunosuppressive agent(s) and/or antibiotic agent(s) may beadministered prior to, during or following the initial application ofthe therapeutic cells and/or pharmaceutical composition(s) of thepresent invention. The intermittent and cyclic administration frameworksprovided herein are exemplary only and not in any way intended to belimiting. Those skilled in the art will recognize various administrationframeworks/frequencies for individual cases, each such administrationframework/frequency is within the scope of the present invention.

Articles and Methods of Manufacture

The present invention also includes an article of manufacture providingtherapeutic cells and/or pharmaceutical composition comprisingtherapeutic cells and/or components of the pharmaceutical composition ofthe present invention for intranasal administration to the upper thirdof the nasal cavity and subsequent bypass of the blood-brain barrier andtransport to the CNS. The article of manufacture may include a vial orother container that contains a composition suitable for the presentmethod together with any carrier, either dried or in liquid form. Thearticle of manufacture further includes instructions in the form of alabel on the container and/or in the form of an insert included in a boxin which the container is packaged, for the carrying out the method ofthe invention. The instructions can also be printed on the box in whichthe vial is packaged. The instructions contain information such assufficient dosage and administration information so as to allow thesubject or a worker in the field to administer the therapeutic cellsand/or pharmaceutical composition comprising therapeutic cells and/orcomponents of the pharmaceutical composition of the present invention.It is anticipated that a worker in the field encompasses any doctor,nurse, technician, spouse, or other caregiver that might administer thetherapeutic cells and/or pharmaceutical composition comprisingtherapeutic cells and/or components of the pharmaceutical composition ofthe present invention. The therapeutic cells and/or the components ofthe pharmaceutical composition may also be self-administered by thesubject.

The present invention may be better understood with reference to thefollowing examples. These examples are intended to be representative ofspecific embodiments of the invention, and are not intended as limitingthe scope of the invention.

EXAMPLES Example 1 Therapeutic Cells Bypassing the Blood-Brain BarrierFollowing Intranasal Application to the Upper Third of the Nasal Cavityin a Rat Model of Parkinson's Disease

The hypothesis that therapeutic cells could indeed bypass theblood-brain barrier was tested by the inventors in healthy rodents (miceand rats) and in rats treated with 6-OHDA to create a model ofParkinson's disease. In this example, mesenchymal stem cells, i.e.,eukaryotic cells, were administered intranasally to the upper third ofthe nasal cavity of adult healthy mice and to 6-hydroxydopamine (6-OHDA)unilateral-lesioned rats to model the damaged and/or degenerating CNS ofpatients with Parkinson's disease. Additionally, glioma cells wereintranasally administered to the upper third of the nasal cavity ofyoung healthy rats. Within one (1) hour of administration/application,both cell types reached the olfactory bulb, cortex, hippocampus,striatum and the cerebellum of the healthy animals. In the 6-OHDA ratmodel of Parkinson's disease, the cells were detected 4 hours afteradministration. It is likely that the cells may have reached the brainin both cases in less than one hour. After the cells crossed thecribriform plate, two migration routes were observed: (1) migration intothe olfactory bulb and also to other parts of the brain including thecortex and striatum; and (2) entry into the cerebrospinal fluid withmovement along the surface of the cortex followed by entrance into thebrain parenchyma.

Example 2 Effect of Delivery-Enhancement Agent on Transport ofTherapeutic Cells Following Intranasal Application to the Upper Third ofNasal Cavity

The efficacy was evaluated of intranasal delivery of therapeutic cellsto the brain after intranasal application of rat mesenchymal stem cells(MSCs) labeled with CFDA or Hoechst dye to the upper third of the nasalcavity of seven-week-old C57 bl/6 mice, thus bypassing the blood-brainbarrier in the administration and application and transport of thetherapeutic cells.

Initially, the animals were divided into three groups (n=5 in eachgroup): 1) group A received only intranasal therapeutic cells; 2) groupB received delivery-enhancement agent hyaluronidase intranasally 30minutes prior to the intranasal application of cells; 3) group Creceived vehicle intranasally (24 μl PBS). One hour after application ofcells, the animals were sacrificed under anesthesia, the skulls werefrozen at −80° C. and sectioned later in sagittal or horizontal slices(20 μm) mounted with medium containing DAPI or PI and analyzed byfluorescent microscopy.

Hoechst dye labeled cells appeared in all layers of the olfactory bulb,striatum, cortex, in the wall and vicinity of the lateral ventricle, andcerebellum of animals in group A, the intranasally delivered therapeuticcells alone. In the olfactory bulb, the cells were distributed over alllayers in animals of group A and B. Intranasal administration ofhyaluronidase (100 U/animal in group B) increased the number of MSCs inthe brain, especially in the olfactory bulb, when compared with thosefrom group A.

The distribution of MSCs in different cortex layers in groups A and Bsuggest migration of therapeutic cells from the surface into theparenchyma. Numerous cells were localized in the subarachnoid space inclose vicinity to MSCs which already reached the upper layer of thecortex. Some of these cells had processes suggesting progress in theirdifferentiation. A large amount of the intranasally-applied CFDA-labeledMSCs remained in the upper nasal cavity (arrows in Figure 2D) 1 h afterapplication indicating that therapeutic cell migration from the nasalmucosa through the cribriform plate into the brain could possiblycontinue for several hours and perhaps even days.

A stepwise migration of cells from the surface of the cortex into thedeeper layers was observed after a certain density of cells is reachedin one layer; aggregates of cells in the deeper layers appear only inthe vicinity of cell rows placed closer to the surface of the cortex.

Example 3 Targeted Migration of Therapeutic Cells to Lesion within CNSFollowing Intranasal Application to the Upper Third of Nasal Cavity

Since the results obtained and described above in Example 2 show that,besides cortex, olfactory bulb and cerebellum, intranasally appliedtherapeutic cells appeared also in the area of striatum, we decided toinvestigate, whether neurodegeneration might target the migration ofapplied cells to the lesion side using a model with a unilateral lesionwith 6-OHDA in adult rats.

Striatal damage was induced in adult rats by unilateral injection (lefthemisphere) of the neurotoxin 6-hydroxydopamine (6-OHDA) to induce aParkinson's type model. The cells were applied in two groups of animals(n=5 in each): 1) without or 2) with intranasal hyaluronidase treatment(200 U/animal) 30 minutes prior to the intranasal administration of thecells three days after the lesion. The brains of animals were withdrawn4 h after application of cells and frozen at −80° C. To show thedegenerative changes in the left (lesioned) striatum after6-OHDA-lesion, 10 horizontal slices from each animal were taken from thearea 5 mm to 8 mm from bregma were stained for tyrosine hydroxylase(TH).

In contrast to the strong staining of nearly whole striatum with TH atthe unlesioned side, the expression of TH at the lesioned side wasclearly decreased. Screening of the brain slices with fluorescentmicroscopy revealed a notable difference in the number of cells betweenthe lesioned and contralateral sides: the majority of CFDA labeled MSCswas found in the olfactory bulb (OB), the cortex at the level of lesionand within the lesioned striatum whereas only very few cells were foundin the striatum, cortex and OB of the contralateral hemisphere. SomeMSCs were occasionally found in the slices stained for TH: Interestinglyvery few of the MSCs which were found in OB expressed TH, whereas themajority of cells localized in the cortex in the vicinity of the lesionwere TH-positive.

These results provide evidence of targeted stem cell preferentialmigration to the site of the lesion in 6-OHDA-lesioned rodents.Furthermore, better delivery to the brain of bone marrow stem cells wasshown in the lesioned hemisphere in comparison with those in theunlesioned side using an embodiment of the present invention.

Example 4 Therapeutic Cells Comprising Tumor Cells Bypassing Blood-BrainBarrier Following Intranasal Application to the Upper Third of NasalCavity in Parkinson's Model

This study investigates whether or not only therapeutic stem cells butalso tumor cells may be delivered to the brain after intranasaladministration. Intranasal administration of human Phi-Yellow andCFDA-labeled T406 glioma cells to the upper third of the nasal cavity of10-day old rats (n=5) was achieved. One hour after administration, theanimals were sacrificed. Sagittal sections (20 μm) of the whole heads ofanimals (including the skull and brain) were processed by fluorescentmicroscopy. CFDA-labeled glioma cells identified in the nasal cavity,cribriform plate, olfactory bulb, frontal cortex, and hippocampal area.

In this study, intranasal delivery of eukaryotic cells (stem cells aswell as tumor cells) into the intact and lesioned brains of rodents wasdemonstrated. Brain tumors consist of intracranial tumors which resultfrom abnormal or uncontrolled cell division. This can occur in thebrain, the meninges, the cranial nerves or in blood vessels orlymphatics of the central nervous system. Most primary brain tumorsoccur in the posterior cranial fossa in children (i.e. brain stemglioma) and in the anterior portion of the cerebral hemispheres inadults. Pediatric brain tumors account for about one-fourth of pediatriccancers. There are about more than 10,000 deaths per year in the UnitedStates due to brain tumors. Most primary brain tumors originate fromglial cells in the central nervous system. However, secondary braintumors that develop from cancers elsewhere in the body and metastasizeto the brain are even more common. Tumors can metastasize to the brainfrom the lungs, skin, kidney, breast, colon and other organs.

Brain tumors are difficult to treat because most chemotherapeutic agentsdo not readily cross the blood-brain barrier and it is not possible tosafely and successfully remove certain types of brain tumors, e.g. brainstem gliomas, because of their location close to areas of the brain thatcontrol key autonomic functions such as breathing, heart function, etc.

Currently, researchers developing and testing new therapeutics for braintumors need to surgically implant tumor cells into the brain of ananimal to create an animal brain tumor model which can be used to testnew drugs. We demonstrate here that tumor cells can be non-invasivelyintroduced into the brain by administering them to the upper third ofthe nasal cavity and that hyaluronidase and other agents can be used tofacilitate this process. Thus this example demonstrates that a braintumor model can be created non-invasively without the problemsassociated with neurosurgery and direct implantation of tumor cellsusing embodiments of the present invention.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

We claim:
 1. A method for treating a damaged or degenerating or injuredcentral nervous system of a mammal, the damage or degeneration caused bya neurological disease or condition that results in the loss or death ofcentral nervous system cells, comprising: applying at least atherapeutically effective amount of therapeutic cells to the upper thirdof the nasal cavity of the mammal; enabling the therapeutic cells toaccess the damaged central nervous system by bypassing the blood-brainbarrier; and thereby treating the mammal's damaged or degenerating orinjured central nervous system.
 2. The method of claim 1, furthercomprising administering the therapeutic cells to a tissue innervated bythe olfactory nerve, wherein the therapeutic cells bypass theblood-brain barrier to access the damaged central nervous system; andminimizing systemic delivery of the therapeutic cells outside of thecentral nervous system.
 3. The method of claim 2, further comprising thetherapeutic cells bypassing the blood-brain barrier by migrating along aneural pathway into the damaged central nervous system.
 4. The method ofclaim 3, further comprising the therapeutic cells preferentiallymigrating to an area of damage within the central nervous system.
 5. Themethod of claim 1, wherein the therapeutic cells comprise eukaryoticcells.
 6. The method of claim 1, wherein the therapeutic cells comprisestem cells.
 7. The method of claim 1, wherein the therapeutic cells arecapable of therapeutic action in the mammal.
 8. The method of claim 1,further comprising applying the therapeutic cells to the upper third ofthe mammal's nasal cavity in a physiologically effective amount toprovide therapeutic action comprising replacement of lost and/or dyingcells in the damaged central nervous system.
 9. The method of claim 1,wherein the neurological disease or condition comprises stroke.
 10. Themethod of claim 1, wherein the neurological disease or conditioncomprises Huntington's disease.
 11. The method of claim 1, wherein theneurological disease or condition comprises Lewy Body Dementia (LBD).12. The method of claim 1, wherein the neurologic disease or conditioncomprises frontotemporal demential (FTD).
 13. The method of claim 1,wherein the neurologic disease or condition comprises progressivesupranuclear palsy (PSP).
 14. The method of claim 1, wherein theneurologic disease or condition comprises corticobasal degeneration(CBD).
 15. The method of claim 1, wherein the neurologic disease orcondition comprises multiple sclerosis.
 16. The method of claim 1,wherein the neurologic disease or condition comprises brain tumors. 17.The method of claim 1, wherein the neurologic disease or conditioncomprises spinal cord injury.
 18. The method of claim 1, wherein theneurologic disease or condition comprises subarachnoid hemorrhage. 19.The method of claim 1, wherein the neurologic disease or conditioncomprises intracerebral hemorrhage.
 20. The method of claim 1, whereinthe neurologic disease or condition comprises traumatic brain injury.